A virus for treatment of tumor

ABSTRACT

Provided are an enterovirus D68 (EV-D68) or a modified form thereof, or a nucleic acid molecule comprising a genomic sequence or cDNA sequence of the EV-D68 or a modified form thereof, or a complementary sequence of the genomic sequence or cDNA sequence, or a pharmaceutical composition comprising the EV-D68 or a modified form thereof, or the nucleic acid molecule, and use of the EV-D68 or a modified form thereof, or the nucleic acid molecule in the manufacture of a pharmaceutical composition for treating a tumor.

TECHNICAL FIELD

The present invention relates to the field of viruses and the field oftumor treatment. Specifically, the present invention relates to use ofan Enterovirus D68 (EV-D68) or a modified form thereof, or a nucleicacid molecule comprising a genomic sequence or cDNA sequence of EV-D68or a modified form thereof, or a complementary sequence of the genomicsequence or cDNA sequence, for treating a tumor in a subject (e g , ahuman), and for manufacture of a medicament for treating a tumor in asubject (e.g., a human). The present invention also relates to a methodfor treating a tumor, which comprises a step of administering to asubject in need thereof EV-D68 or a modified form thereof, or a nucleicacid molecule comprising a genomic sequence or cDNA sequence of EV-D68or a modified form thereof, or a complementary sequence of the genomicsequence or cDNA sequence.

BACKGROUND ART

The current methods for treatment of malignant tumors include surgerychemotherapy and radiotherapy. These traditional therapies are notsatisfactory for the treatment of metastatic tumors, and may also causegreat harm to patients' health. In contrast, as a new type of treatmentmethod, the tumor treatment method using oncolytic virus has highspecificity, good effectiveness, and less side effects, and is currentlyconsidered as a promising tumor treatment method.

Oncolytic virus is a virus that can self-replicate in tumor cells,thereby killing and lysing the tumor cells, or arresting the growth ofthe tumor cells. When used in in vivo treatment, oncolytic virus showsspecificity for tumor cells, and can directly induce death of tumorcells, but has little or no effect on normal cells. Meanwhile, oncolyticvirus can also induce cytotoxic T lymphocyte response in the immunesystem, thereby indirectly killing tumor cells.

Enterovirus belongs to Picornaviridae family, and its genome issingle-stranded positive-sense RNA. There are following advantages inusing enterovirus as oncolytic virus: firstly, as single-stranded RNAvirus, its genome won't undergo any stages of DNA in the host, so thatthere won't be genotoxicity caused by the insertion of the viral genomeinto the host's DNA, which has better safety; secondly, the enterovirusgenome is relatively small, and a large number of viruses can bereplicated in a short period of time to further infect other tumorcells, causing a strong cytopathic effect; next, the enterovirus doesnot contain oncogenes and therefore does not induce tumor; and finally,the genome of the enterovirus can be modified by reverse geneticstechnology to achieve attenuation of virus and reduce its side effects.

At present, the reported enteroviruses with oncolytic activity includechimeric polioviruses for the treatment of human solid tumors such asmalignant gliomas (Dobrikova et al., Mol Ther 2008, 16 (11): 1865-1872);Coxsackie viruses A13, A15, A18, and A21 that kill human melanoma cells(Au et al., \Tirol J 2011, 8: 22); Echo virus ECHO1 that kills humangastric cancer cells and ovarian cancer cells (Shafren et al., Int JCancer 2005, 115 (2): 320-328; Haley et al., J Mol Med (Berl) 2009, 87(4): 385-399) and the like. However, it is still necessary to obtainviruses with both tumor-specificity and tumor-killing activity.

Enterovirus D68 (EV-D68) is a kind of Enterovirus D of the genusEnteroviruses of Picornaviridae family, which was first isolated fromchildren with respiratory infections in California in 1962 (Schieble etal., Am J Epidemiol 1967, 85 (2): 297-310). Unlike most enteroviruses,which are resistant to acids and reproduce in the human gastrointestinaltract, EV-D68 is sensitive to acids and replicates mainly in therespiratory tract. Since there have been few reports of EV-D68 infectionfor a long time, EV-D68 is considered to be a rare pathogen that mainlycauses mild respiratory diseases, including ninny nose, sneezing, andcough. At present, there is not report in the art that enterovirus 68has oncolytic activity.

CONTENTS OF THE INVENTION

After a lot of experiments and repeated explorations, it is unexpectedlyfound that Enterovirus D68 has a broad spectrum and significant tumorcell killing ability. Based on this finding, the inventors of thepresent invention have developed a new oncolytic virus for treatingtumors and a tumor treatment method based on the virus.

Medical use

In a first aspect, the present invention provides use of an EnterovirusD68 (EV-D68) or a modified form thereof, or an isolated nucleic acidmolecule, in treatment of a tumor in a subject, or in the manufacture ofa medicament for treating a tumor in a subject; wherein the isolatednucleic acid molecule comprises a sequence selected from the following:

-   -   (1) a genomic sequence or cDNA sequence of EV-D68 or a modified        form thereof; and    -   (2) a complementary sequence of the genomic sequence or cDNA        sequence. In certain preferred embodiments, the EV-D68 is a        wild-type EV-D68. In certain preferred embodiments, the EV-D68        may be a clinical isolate isolated from an individual infected        with the Enterovirus D68.

In certain preferred embodiments, the EV-D68 or a modified form thereofhas a genomic sequence that has a sequence identity of at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% to a nucleotide sequence as shown inSEQ ID NO: 12. In certain preferred embodiments, the genomic sequence ofthe EV-D68 or a modified form thereof is a nucleotide sequence as shownin SEQ ID NO: 12.

In certain preferred embodiments, the EV-D68 or a modified form thereofhas a cDNA sequence that has a sequence identity of at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% to a nucleotide sequence shown in SEQID NO: 1. In certain preferred embodiments, the cDNA sequence of theEV-D68 or a modified form thereof is a nucleotide sequence as shown inSEQ ID NO: 1.

In certain preferred embodiments, the modified form is a modifiedEV-D68, which has a substitution, insertion, or deletion of one or morenucleotides in the genome as compared to a wild-type EV-D68.

In certain preferred embodiments, as compared to the wild-type EV-D68,the modified EV-D68 has one or more modifications selected from thefollowing:

-   -   (1) one or more mutations in an untranslated region (e.g., 5′UTR        or 3′UTR);    -   (2) an insertion of one or more exogenous nucleic acids;    -   (3) a deletion or mutation of one or more endogenous genes; and    -   (4) any combination of the above three items.

In certain preferred embodiments, the modified EV-D68 includes one ormore mutations in the 5′ untranslated region (5′UTR).

In certain preferred embodiments, the modified EV-D68 has a substitutionof all or part of the 5′UTR sequence. In certain preferred embodiments,the internal ribosome entry site (IRES) sequence in the 5′UTR of themodified EV-D68 is replaced with an exogenous IRES sequence, such as theinternal ribosome entry site sequence of human rhinovirus 2 (HRV2). Incertain preferred embodiments, the internal ribosome entry site sequenceof the human rhinovirus 2 (HRV2) is shown in SEQ ID NO: 2.

The use of the internal ribosome entry site sequence of human rhinovirus2 (HRV2) is advantageous in some cases, for example, it is conducive toimprovement of the tumor specificity of oncolytic viruses. It has beenpreviously reported that in normal human nerve cells, the internalribosome entry site sequence of human rhinovirus 2 is specifically boundby host RNA-binding proteins (DRBP76 and NF45), thereby preventing therecruitment of factors such as e1F4G (Merrill et al. J Virol 2006, 80(7): 3147-3156; Merrill and Gromeier, J Virol 2006, 80 (14): 6936-6942;Neplioueva et al., PLoS One 2010, 5 (7): e11710); meanwhile, due to thelack of support of signaling pathways such as Raf/Erk1/2/MAPK, it isdifficult for ribosomes to bind to the internal ribosome entry sitesequence of human rhinovirus 2, so that it is impossible to initiatetranslation of viral protein (Dobrikov et al., Mol Cell Biol 2011,31(14): 2947-2959; Dobrikov et al., Mol Cell Biol 2013, 33(5): 937-946).In human glioma tumor cells, the internal ribosome entry site of humanrhinovirus 2 is not affected by the above two factors, and thus cannormally initiate transcription and translation of viral protein.Therefore, in some cases, replacing the internal ribosome entry sitesequence of EV-D68 with the internal ribosome entry site sequence ofhuman rhinovirus 2 is beneficial to avoid or reduce the toxic and sideeffect of the virus of the present invention to normal human nervecells, without affecting the use of the virus in the treatment of humangliomas.

In certain preferred embodiments, the modified EV-D68 comprises anexogenous nucleic acid.

In certain preferred embodiments, the exogenous nucleic acid encodes acytokine (e.g., GM-CSF, preferably human GM-CSF), or an antitumorprotein or polypeptide (e.g., a scFv against PD-1 or PD-L1, preferably ascFv against human PD-1 or PD-L1). In certain preferred embodiments, theexogenous nucleic acid is inserted between the 5′UTR gene and the VP4gene, or between the VP1 gene and the 2A gene of the genome of themodified EV-D68.

In certain preferred embodiments, the exogenous nucleic acid comprises atarget sequence of microRNA (miRNA) (e.g., miR-133 or miR-206). Incertain preferred embodiments, the target sequence of microRNA insertedin the 3′ untranslated region (3′UTR) of the genome of the modifiedEV-D68.

It has been previously reported that the expression level of certainmicroRNA in tumor cells is significantly lower than normal cells and/orhas obvious tissue specificity. Therefore, in some cases, the modifiedEV-D68 of the present invention containing a target sequence of suchmicroRNA is advantageous, because such microRNA that are highlyexpressed in normal cells or tissues can reduce or even block thereplication of the modified EV-D68 in the normal cells or tissues by thecorresponding target sequence, thereby reducing even avoiding the toxicside effects of the modified EV-D68 on non-tumor cells. Such microRNAsinclude but are not limited to miR-133, miR-206, miR-1, miR-143,miR-145, miR-217, let-7, miR-15, miR-16, etc. (see, for example, PCTInternational Application WO2008103755A1, US patent applicationUS20160143969A1, or Baohong Zhang et al., Developmental Biology, Volume302, Issue 1, 1 Feb. 2007, Pages 1-12; all of these documents areincorporated herein by reference).

In certain preferred embodiments, the exogenous nucleic acid comprises atarget sequence of one or more (e.g., 2, 3, or 4) microRNA as describedabove. In certain preferred embodiments, the exogenous nucleic acidcomprises a target sequence of miR-133 and/or miR-206. In certainpreferred embodiments, the target sequence of miR-133 is shown in SEQ IDNO: 3. In certain preferred embodiments, the target sequence of miR-206is shown in SEQ ID NO: 4. In some cases, the insertion of the targetsequence of miR-133 and/or miR-206 is advantageous. This is becausemiR-133 and miR-206 are specifically expressed in muscle tissue, so thatthe tissue tropism of the oncolytic virus can be changed by insertingthe target sequence of miR-133 and/or miR-206 into the modified EV-D68,thereby reducing or avoiding damage to normal muscle tissue.

In certain preferred embodiments, the modified EV-D68 comprises at leastone insertion of the exogenous nucleic acid as described above and/or atleast one mutation in the untranslated region as described above.

In certain preferred embodiments, the genomic sequence of the modifiedEV-D68 has a sequence identity of at least 70%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% to a nucleotide sequence selected from: the nucleotidesequences as shown in SEQ ID NOs: 13-16. In certain preferredembodiments, the genomic sequence of the modified EV-D68 is selectedfrom the nucleotide sequences as shown in any one of SEQ ID NOs: 13-16.

In certain preferred embodiments, the cDNA sequence of the modifiedEV-D68 has a sequence identity of at least 70%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% to a nucleotide sequence selected from: the nucleotidesequences as shown in SEQ ID NOs: 8-11. In certain preferredembodiments, the cDNA sequence of the modified EV-D68 is selected fromthe nucleotide sequences as shown in any one of SEQ ID NOs: 8-11.

In the present invention, the modified EV-D68 can be obtained by reversegenetics technology, and the reverse genetics technology is known in theart, for example, see Yang L S, Li S X, Liu Y J, et al Virus Res, 2015,210: 165-168; Hou W H, Yang L S, Li S X, et al. Virus Res, 2015, 205:41-44; which is incorporated herein by reference in its entirety. Insuch embodiments, the modified EV-D68 is typically obtained by modifyingthe cDNA of wild-type EV-D68 (e.g., insertion of an exogenous nucleicacid, deletion or mutation of an endogenous gene, or mutation in anon-translated region).

In the present invention, the EV-D68 or a modified form thereof may bepretreated to reduce or eliminate the immune response against the virusin a subject, wherein the pretreatment may comprise: packaging theEV-D68 in a lipidosome or micelle, and/or using a protease (e.g.,chymotrypsin or trypsin) to remove the capsid protein of the virus toreduce the humoral and/or cellular immunity against the virus in host.

In the present invention, the EV-D68 or a modified form thereof can beserially passaged for adaptation in tumor cells. In certain preferredembodiments, the tumor cells may be tumor cell lines or tumor cellstrains known in the art, or may be tumor cells obtained by surgicalresection or clinical isolation from an individual (e.g., a subject)having a tumor. In certain preferred embodiments, the EV-D68 or amodified form thereof is serially passaged for adaptation in tumor cellsobtained from an individual (e.g., a subject) having a tumor. In certainpreferred embodiments, the tumor cells are obtained by surgicalresection or clinical isolation from an individual (e.g., a subject)having a tumor. In certain preferred embodiments, the method for serialpassaging for adaptation comprises a plurality of (e.g., at least 5, atleast 10, at least 15, at least 20) cycles consisting of the followingprocesses: 1) infecting a target tumor cell with a virus; 2) harvestingthe virus in a supernatant; and 3) reinfecting a fresh target tumor cellwith the obtained virus.

In certain preferred embodiments, the EV-D68 and modified forms thereofas described above can be used in combination. Therefore, the medicamentmay comprise one or several of EV-D68 and modified forms thereof.

In certain preferred embodiments, the isolated nucleic acid moleculeconsists of a genomic sequence or cDNA sequence of EV-D68 or a modifiedform thereof as described above, or a complementary sequence of thegenomic sequence or cDNA sequence. In certain preferred embodiments, theisolated nucleic acid molecule has a genomic sequence of EV-D68 or amodified form thereof as described above. In certain preferredembodiments, the isolated nucleic acid molecule is RNA. In certainpreferred embodiments, the isolated nucleic acid molecule has anucleotide sequence as shown in any one of SEQ ID NOs: 12-16.

In certain preferred embodiments, the isolated nucleic acid molecule isa vector (e.g. cloning vector or expression vector) comprising a genomicsequence or cDNA sequence of EV-D68 or a modified form thereof asdescribed above, or a complementary sequence of the genomic sequence orcDNA sequence. In certain preferred embodiments, the isolated nucleicacid molecule is a vector (e.g., cloning vector or expression vector)comprising a cDNA sequence of EV-D68 or a modified form thereof asdescribed above, or a complementary sequence of the cDNA sequence. Incertain preferred embodiments, the isolated nucleic acid molecule is avector comprising a nucleotide sequence as shown in any one of SEQ IDNOs: 1, 8-11 or a complementary sequence thereof.

In certain preferred embodiments, the isolated nucleic acid moleculecomprises the complementary sequence of the genomic sequence of EV-D68or a modified form thereof as described above. In certain preferredembodiments, the complementary sequence is complementary to a nucleotidesequence selected from:

(1) a nucleotide sequence as shown in SEQ ID NO: 12;

(2) a nucleotide sequence having a sequence identity of at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% to the nucleotide sequence as shown inSEQ ID NO: 12;

(3) a nucleotide sequence as shown in any one of SEQ ID NOs: 13-16; and

(4) a nucleotide sequence having a sequence identity of at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% to the nucleotide sequence as shown inany of SEQ ID NOs: 13-16.

In certain preferred embodiments, the isolated nucleic acid moleculecomprises a complementary sequence to the cDNA sequence of EV-D68 or amodified form thereof as described above. In certain preferredembodiments, the complementary sequence is complementary to a nucleotidesequence selected from:

(1) a nucleotide sequence as shown in SEQ ID NO: 1;

(2) a nucleotide sequence having a sequence identity of at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% to the nucleotide sequence as shown inSEQ ID NO: 1;

(3) a nucleotide sequence as shown in any one of SEQ ID NOs: 8-11; and

(4) a nucleotide sequence having a sequence identity of at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% to the nucleotide sequence as shown inany one of SEQ ID NOs: 8-11.

In the present invention, the isolated nucleic acid molecule can bedelivered by any means known in the art, for example, a naked nucleicacid molecule (e.g., a naked RNA) can be directly injected, or anon-viral delivery system can be used. The non-viral delivery system canbe obtained from a variety of materials well known in the art,including, but not limited to, the materials described in detail in “YinH, et al. Nat Rev Genet. 2014 August; 15(8): 541-55.” and “Riley M K,Vermerris W. Nanomaterials (Base1). 2017 Apr. 28; 7(5). Pii: E94.”,which are incorporated herein by reference in their entirety, such asliposomes, inorganic nanoparticles (such as gold nanoparticles),polymers (such as PEG), and so on.

In certain preferred embodiments, the medicament comprises atherapeutically effective amount of the EV-D68 and/or a modified formthereof as described above, or a therapeutically effective amount of theisolated nucleic acid molecule as described above. In certain preferredembodiments, the medicament may be in any form known in the medicalarts. For example, the medicament may be in the form of a tablet, apill, a suspension, an emulsion, a solution, a gel, a capsule, a powder,a granule, an elixir, a lozenge, a suppository, or an injection(including injection solution, lyophilized powder) and so on. In someembodiments, the medicament is an injection solution or a lyophilizedpowder.

In certain preferred embodiments, the medicament further comprises apharmaceutically acceptable carrier or excipient. In certain preferredembodiments, the medicament comprises a stabilizer.

In certain preferred embodiments, the medicament optionally furthercomprises an additional pharmaceutically active agent. in a preferredembodiment, the additional pharmaceutically active agent is a medicamenthaving antitumor activity, such as an additional oncolytic virus,chemotherapeutic agent or immunotherapeutic agent.

In the present invention, the additional oncolytic virus includes, butis not limited to, herpesvirus, adenovirus, parvovirus, reovirus,Newcastle disease virus, vesicular stomatitis virus, measles virus, orany combination thereof. The chemotherapeutic agent includes but is notlimited to 5-fluorouracil, mitomycin, methotrexate, hydroxyurea,cyclophosphamide, dacarbazine, mitoxantrone, anthracyclines (e.g.,epirubicin or doxorubicin), etoposide, platinum compounds (e.g.,carboplatin or cisplatin), taxanes (e.g., paclitaxel or taxotere), orany combination thereof. The immunotherapeutic agent includes, but isnot limited to, immune checkpoint inhibitors (e.g., PD-L1/PD-1inhibitors or CTLA-4 inhibitors), tumor-specific targeting antibodies(e.g., rituximab or Herceptin) or any combination thereof.

In certain preferred embodiments, the medicament comprises a unit doseof the EV-D68 and/or a modified form thereof as described above, forexample comprising at least 1×10² pfu, at least 1×10³ pfu, at least1×10⁴ pfu, 1×10⁵ pfu, 1×10⁶ pfu, at least 1×10⁷ pfu, at least 1×10⁸ pfu,at least 1×10⁹ pfu, at least 1×10¹° pfu, at least 1×10¹¹ pfu, at least1×10¹² pfu, at least 1×10¹³ pfu, at least 1×10¹⁴ pfu, or at least 1×10¹⁶pfu of the EV-D68 and/or a modified form thereof. In certain preferredembodiments, the medicament comprises 1×10² pfu to 1×10¹⁷ pfu of theEV-D68 and/or a modified form thereof as described above.

In certain preferred embodiments, the medicament contains a unit dose ofan isolated nucleic acid molecule as described above, such as thenucleic acid molecule containing 3×10¹⁰ to 3×10¹⁴ virus genome copies.

In certain preferred embodiments, the medicament may be administered incombination with an additional therapy. This additional therapy may beany therapy known for tumors, such as surgery, chemotherapy, radiationtherapy, immunotherapy, hormone therapy or gene therapy. This additionaltherapy may be administered before, concurrently with, or after theadministration of the medicament.

In certain preferred embodiments, the tumor includes, but is not limitedto, cervical cancer, ovarian cancer, endometrial cancer, lung cancer,liver cancer, kidney cancer, neuroblastoma, glioma, breast cancer,melanoma, prostate cancer, bladder cancer, pancreatic cancer, gastriccancer, colorectal cancer, esophageal cancer, thyroid cancer, laryngealcancer, osteosarcoma, hematopoietic malignancy (e.g., lymphoma orleukemia).

In certain preferred embodiments, the subject is a mammal, such as ahuman.

In another aspect, the invention also relates to use of the EV-D68and/or a modified form thereof as defined in the first aspect, or theisolated nucleic acid molecule as defined in the first aspect, as amedicament.

Treatment Method

In a second aspect, the present invention provides a method for treatinga tumor, comprising the step of administering to a subject in needthereof an effective amount of an EV-D68 or a modified form thereof, oran effective amount of an isolated nucleic acid molecule; wherein theisolated nucleic acid molecule comprises a sequence selected from thegroup consisting of:

(1) a genomic sequence or cDNA sequence of EV-D68 or a modified formthereof; and

(2) a complementary sequence of the genomic sequence or cDNA sequence.

In certain preferred embodiments, EV-D68 is administered to the subject.In certain preferred embodiments, the EV-D68 is wild-type EV-D68. Incertain preferred embodiments, the EV-D68 may be a clinical isolate thatis isolated from an individual infected with Enterovirus D68

In certain preferred embodiments, the genomic sequence of the EV-D68 ora modified form thereof has a sequence identity of at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% to the nucleotide sequence as shown inSEQ ID NO: 12. In certain preferred embodiments, the genomic sequence ofthe EV-D68 or a modified form thereof is a nucleotide sequence as shownin SEQ ID NO: 12.

In certain preferred embodiments, the cDNA sequence of the EV-D68 or amodified form thereof has a sequence identity of at least 70%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% to the nucleotide sequence as shown in SEQ IDNO: 1. In certain preferred embodiments, the cDNA sequence of the EV-D68or a modified form thereof is a nucleotide sequence as shown in SEQ IDNO: 1.

In certain preferred embodiments, a modified form of EV-D68 isadministered to the subject. In certain preferred embodiments, ascompared to the wild-type EV-D68, the modified form is a modifiedEV-D68, which has a substitution, insertion, or deletion of one or morenucleotides in the genome.

In certain preferred embodiments, as compared to the wild-type EV-D68,the modified EV-D68 has one or more modifications selected from thefollowing:

(1) one or more mutations in an untranslated region (e.g., 5′UTR or3′UTR);

(2) an insertion of one or more exogenous nucleic acids;

(3) a deletion or mutation of one or more endogenous genes; and

(4) any combination of the above three items.

In certain preferred embodiments, the modified EV-D68 includes one ormore mutations in the 5′ untranslated region (5′UTR).

In certain preferred embodiments, the modified EV-D68 has a substitutionof all or part of the 5′UTR sequence. In certain preferred embodiments,the internal ribosome entry site (IRES) sequence in the 5′UTR of themodified EV-D68 is replaced with an exogenous IRES sequence, such as theinterior ribosome entry site sequence of human rhinovirus 2 (HRV2). Incertain preferred embodiments, the internal ribosome entry site sequenceof the human rhinovirus 2 (HRV2) is shown in SEQ ID NO: 2.

In certain preferred embodiments, the modified EV-D68 comprises anexogenous nucleic acid.

In certain preferred embodiments, the exogenous nucleic acid encodes acytokine (e.g., GM-CSF, preferably human GM-CSF), or an antitumorprotein or polypeptide (e.g., scFv against PD-1 or PD-L1, preferablyscFv against human PD-1 or PD-L1). In certain preferred embodiments, theexogenous nucleic acid is inserted between the 5′UTR gene and the VP4gene, or between the VP1 gene and the 2A gene of the genome of themodified EV-D68.

In certain preferred embodiments, the exogenous nucleic acid comprises atarget sequence of microRNA (miRNA) (e.g., miR-133 or miR-206) Incertain preferred embodiments, the target sequence of microRNA isinserted in the 3′ untranslated region (3′UTR) of the genome of themodified EV-D68.

In certain preferred embodiments, the exogenous nucleic acid comprises atarget sequence of one or more (e.g., 2, 3, or 4) microRNA as describedabove. In certain preferred embodiments, the exogenous nucleic acidcomprises a target sequence of miR-133 and/or miR-206. In certainpreferred embodiments, the target sequence of miR-133 is shown in SEQ IDNO: 3. In certain preferred embodiments, the target sequence of miR-206is shown in SEQ ID NO: 4.

In certain preferred embodiments, the modified EV-D68 comprises at leastone insertion of the exogenous nucleic acid as described above and/or atleast one mutation in the untranslated region as described above.

In certain preferred embodiments, the genomic sequence of the modifiedEV-D68 has a sequence identity of at least 70%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95° A, at least 96%, at least 97%, at least 98%, at least99%, or 100% to a nucleotide sequence selected from: the nucleotidesequences as shown in SEQ ID NOs: 13-16. In certain preferredembodiments, the genomic sequence of the modified EV-D68 is selectedfrom the nucleotide sequence as shown in any one of SEQ ID NOs: 13-16.

In certain preferred embodiments, the cDNA sequence of the modifiedEV-D68 has a sequence identity of at least 70%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% to a nucleotide sequence selected from: the nucleotidesequences as shown in SEQ ID NOs: 8-11. In certain preferredembodiments, the cDNA sequence of the modified EV-D68 is selected fromthe nucleotide sequence as shown in any one of SEQ ID NOs: 8-11.

In certain preferred embodiments, the EV-D68 and modified forms thereofas described above can be used in combination. Thus, one or more of theEV-D68 and modified forms can be administered to a subject.

In certain preferred embodiments, the isolated nucleic acid molecule asdescribed above is administered to the subject.

In certain preferred embodiments, the isolated nucleic acid moleculeconsists of the genomic sequence or cDNA sequence of the EV-D68 or amodified form thereof as described above, or the complementary sequenceof the genomic sequence or cDNA sequence. In certain preferredembodiments, the isolated nucleic acid molecule has the genomic sequenceof the EV-D68 or a modified form thereof as described above. In certainpreferred embodiments, the isolated nucleic acid molecule is RNA. Incertain preferred embodiments, the isolated nucleic acid molecule has anucleotide sequence as shown in any one of SEQ ID NOs: 12-16.

In certain preferred embodiments, the isolated nucleic acid molecule isa vector (e.g. cloning vector or expression vector) comprising thegenomic sequence or cDNA sequence of EV-D68 or a modified form thereofas described above, or the complementary sequence of the genomicsequence or cDNA sequence. In certain preferred embodiments, theisolated nucleic acid molecule is a vector (e.g., cloning vector orexpression vector) comprising the cDNA sequence of EV-D68 or a modifiedform thereof as described above, or the complementary sequence of thecDNA sequence. In certain preferred embodiments, the isolated nucleicacid molecule is a vector comprising the nucleotide sequence as shown inany one of SEQ ID NOs: 1, 8-11 or the complementary sequence thereof.

In certain preferred embodiments, the isolated nucleic acid moleculecomprises the complementary sequence of the genomic sequence of EV-D68or a modified form thereof as described above. In certain preferredembodiments, the complementary sequence is complementary to a nucleotidesequence selected from:

(1) a nucleotide sequence as shown in SEQ ID NO: 12;

(2) a nucleotide sequence having a sequence identity of at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% to the nucleotide sequence as shown inSEQ ID NO: 12;

(3) a nucleotide sequence as shown in any one of SEQ ID NOs: 13-16; and

(4) a nucleotide sequence having a sequence identity of at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% to the nucleotide sequence shown in anyof SEQ ID NOs: 13-16.

In certain preferred embodiments, the isolated nucleic acid moleculecomprises the complementary sequence of the cDNA sequence of EV-D68 or amodified form thereof as described above. In certain preferredembodiments, the complementary sequence is complementary to a nucleotidesequence selected from:

(1) a nucleotide sequence as shown in SEQ ID NO: 1;

(2) a nucleotide sequence having a sequence identity of at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% to the nucleotide sequence as shown inSEQ ID NO: 1;

(3) a nucleotide sequence as shown in any one of SEQ ID NOs: 8-11; and

(4) a nucleotide sequence having a sequence identity of at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% to the nucleotide sequence as shown inany one of SEQ ID NOs: 8-11.

In the present invention, the isolated nucleic acid molecule can bedelivered by any means known in the art, for example, a naked nucleicacid molecule (e.g., naked RNA) can be directly injected, or a non-viraldelivery system can be used. The non-viral delivery system can beobtained from a variety of materials well known in the art, including,but not limited to, the materials described in detail in “Yin H, et al.Nat Rev Genet. 2014 August; 15(8): 541-55.” and “Riley M K, Vermerris W.Nanomaterials (Base1). 2017 Apr. 28; 7(5). Pii: E94.”, which areincorporated herein by reference in their entirety, such as liposomes,inorganic nanoparticles (such as gold nanoparticles), polymers (such asPEG), and so on.

In certain preferred embodiments, the EV-D68 and/or a modified formthereof as described above, or the isolated nucleic acid molecule asdescribed above, can be formulated and administered as a pharmaceuticalcomposition. Such a pharmaceutical composition may comprise atherapeutically effective amount of the EV-D68 and/or a modified formthereof as described above, or a therapeutically effective amount of theisolated nucleic acid molecule as described above. In certain preferredembodiments, the pharmaceutical composition may be in any form known inthe medical arts. For example, the pharmaceutical composition may be inthe form of a tablet, a pill, a suspension, an emulsion, a solution, agel, a capsule, a powder, a granule, an elixir, a lozenge, asuppository, or an injection (including injection solution, lyophilizedpowder) and so on. In some embodiments, the medicament is an injectionsolution or a lyophilized powder.

In certain preferred embodiments, the pharmaceutical composition furthercomprises a pharmaceutically acceptable carrier or excipient. In certainpreferred embodiments, the pharmaceutical composition comprises astabilizer.

In the present invention, the EV-D68 and/or a modified form thereof, orthe isolated nucleic acid molecule as described above can beadministered to a subject by any suitable administration route. In somecases, the route of administration of the EV-D68 and/or a modified formthereof, or the isolated nucleic acid molecules as described above,depends on the location and type of tumor. For example, for a solidtumor that is easily accessible, the virus or nucleic acid molecule isoptionally administered by injection directly into the tumor (e.g.,intratumoral injection); for a tumor of hematopoietic system, the virusor nucleic acid molecule can be administered by intravenous or otherintravascular routes; for a tumor that is not easily accessible in thebody (e.g., metastases), the virus or nucleic acid molecule can beadministered systematically so that it can run over the whole body andthereby reaching the tumor (e.g., intravenous or intramuscularinjection). Optionally, the virus or nucleic acid molecule of thepresent invention can be administrated via subcutaneous,intraperitoneal, intrathecal (e.g., for brain tumors), topical (e.g.,for melanoma), oral (e.g., for oral or esophageal cancer), intranasal orinhalation spray (e.g., for lung cancer) routes and so on. In certainpreferred embodiments, the EV-D68 and/or a modified form thereof asdescribed above, or the isolated nucleic acid as described above, can beadministered via intradermal, subcutaneous, intramuscular, intravenous,oral routes etc.

In certain preferred embodiments, the method further comprisesadministering an additional pharmaceutically active agent havingantitumor activity. This additional pharmaceutically active agent may beadministered before, concurrently with or after the administration ofthe EV-D68 and/or a modified form thereof, or an isolated nucleic acidmolecule as described above.

In certain preferred embodiments, the additional pharmaceutically activeagent includes an additional oncolytic virus, chemotherapeutic agent, orimmunotherapeutic agent. In the present invention, the additionaloncolytic virus includes, but is not limited to, herpesvirus,adenovirus, parvovirus, reovirus, Newcastle disease virus, vesicularstomatitis virus, measles virus, or any combination thereof. Thechemotherapeutic agent includes but is not limited to 5-fluorouracil,mitomycin, methotrexate, hydroxyurea, cyclophosphamide, dacarbazine,mitoxantrone, anthracyclines (such as epirubicin or doxorubicin),etoposide, platinum compounds (such as carboplatin or cisplatin),taxanes (such as paclitaxel or taxotere), or any combination thereof.The immunotherapeutic agents include, but are not limited to, immunecheck point inhibitors (such as PD-L1/PD-1 inhibitors or CTLA-4inhibitors), tumor-specific targeting antibodies (such as rituximab orHerceptin) or any combination thereof.

In certain preferred embodiments, the EV-D68 and/or a modified formthereof can be administered in any amount from 1 to 1×10¹⁵ pfu/kg of thesubject's body weight, for example, the EV-D68 and/or a modified formthereof is administered in an amount of at least 1×10³ pfu/kg, at least1×10⁴ pfu/kg, 1×10⁵ pfu/kg, 1×10⁶ pfu/kg, at least 1×10⁷ pfu/kg, atleast 1×10⁸ pfu/kg, at least 1×10⁹ pfu/kg, at least 1×10¹⁰ pfu/kg, atleast 1×10¹¹ pfu/kg, or at least 1×10¹² pfu/kg of the subjects bodyweight. In certain preferred embodiments, the isolated nucleic acidmolecule as described above can be administered in any amount of 3×10¹⁰to 3×10¹⁴ virus genome copies per kg of the subject's body weight. Incertain preferred embodiments, the EV-D68 and/or a modified form thereofor the isolated nucleic acid molecule as described above can beadministered 3 times a day, 2 times a day, 1 time a day, once every 2days or once a week, optionally the above dosage regimen can be repeatedweekly or monthly as appropriate.

In certain preferred embodiments, the method further comprisesadministering an additional therapy. This additional therapy may be anytherapy known for tumors, such as surgery, chemotherapy, radiationtherapy, immunotherapy, hormone therapy or gene therapy. This additionaltherapy may be administered before, concurrently with, or after theadministration of the method described above.

In certain preferred embodiments, the subject is a mammal, such as ahuman.

In certain preferred embodiments, the tumor includes, but is not limitedto, cervical cancer, ovarian cancer, endometrial cancer, lung cancer,liver cancer, kidney cancer, neuroblastoma, glioma, breast cancer,melanoma, prostate cancer, bladder cancer, pancreatic cancer, gastriccancer, colorectal cancer, esophageal cancer, thyroid cancer, laryngealcancer, osteosarcoma, hematopoietic malignancy (e.g., lymphoma orleukemia).

Pharmaceutical Composition

In a third aspect, the present invention provides a pharmaceuticalcomposition comprising an EV-D68 and/or a modified form thereof asdefined in the first or second aspect, or an isolated nucleic acidmolecule as defined in the first or second aspect.

In certain preferred embodiments, the pharmaceutical compositioncomprises the EV-D68 and/or a modified form thereof as defined in thefirst or second aspect. In certain preferred embodiments, the EV-D68and/or modified forms thereof may be used in combination. Therefore, thepharmaceutical composition of the present invention may comprise one orseveral of the EV-D68 and/or modified forms thereof. In certainpreferred embodiments, the pharmaceutical composition comprises a unitdose of the EV-D68 and/or a modified form thereof, for example at least1×10² pfu, at least 1×10³ pfu, at least 1×10⁴ pfu, 1×10⁵ pfu, 1×10⁶ pfu,at least 1×10⁷ pfu, at least 1×10⁸ pfu, at least 1×10⁹ pfu, at least1×10¹⁰ pfu, at least 1×10¹¹ pfu, at least 1×10¹² pfu, at least 1×10¹³pfu, at least 1×10¹⁴ pfu, or at least 1×10¹⁶ pfu of the EV-D68 and/or amodified form thereof. In certain preferred embodiments, thepharmaceutical composition comprises 1×10² pfu to 1×10¹⁷ pfu of theEV-D68 and/or a modified form thereof.

In certain preferred embodiments, the pharmaceutical compositioncomprises an isolated nucleic acid molecule as defined in the firstaspect or the second aspect. In certain preferred embodiments, theisolated nucleic acid molecules can be used in combination. Therefore,the pharmaceutical composition of the present invention may include oneor several of the isolated nucleic acid molecules. In certain preferredembodiments, the pharmaceutical composition comprises a unit dose of theisolated nucleic acid molecule, for example 3×10¹⁰ to 3×10¹⁴ virusgenome copies of the isolated nucleic acid molecule.

In certain preferred embodiments, the pharmaceutical composition may bein any form known in the medical arts. For example, the pharmaceuticalcomposition may be in the form of a tablet, a pill, a suspension, anemulsion, a solution, a gel, a capsule, a powder, a granule, an elixir,a lozenge, a suppository, or an injection (including injection solution,lyophilized powder) and so on. In some embodiments, the medicament is aninjection solution or a lyophilized powder.

In certain preferred embodiments, the pharmaceutical composition furthercomprises a pharmaceutically acceptable carrier or excipient. In certainpreferred embodiments, the pharmaceutical composition comprises astabilizer.

In certain preferred embodiments, the pharmaceutical compositionoptionally further comprises an additional pharmaceutically activeagent. In a preferred embodiment, the additional pharmaceutically activeagent is a medicament having antitumor activity, such as an additionaloncolytic virus, chemotherapeutic agent or immunotherapeutic agent.

In certain preferred embodiments, the pharmaceutical composition is usedto treat a tumor in a subject.

In certain preferred embodiments, the subject is a mammal, such as ahuman.

In certain preferred embodiments, the tumor includes, but is not limitedto, cervical cancer, ovarian cancer, endometrial cancer, lung cancer,liver cancer, kidney cancer, neuroblastoma, glioma, breast cancer,melanoma, prostate cancer, bladder cancer, pancreatic cancer, gastriccancer, colorectal cancer, esophageal cancer, thyroid cancer, laryngealcancer, osteosarcoma, hematopoietic malignancy (e.g., lymphoma orleukemia).

Modified EV-D68

In a fourth aspect, the present invention provides a modified EV-D68having a substitution, insertion, or deletion of one or more nucleotidesin the genome compared to wild-type EV-D68.

In certain preferred embodiments, the genomic sequence of the wild-typeEV-D68 has a sequence identity of at least 70%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% to a nucleotide sequence selected from the nucleotidesequence as shown in SEQ ID NO: 12. In certain preferred embodiments,the genomic sequence of the wild-type EV-D68 is a nucleotide sequence asshown in SEQ ID NO: 12.

In certain preferred embodiments, the cDNA sequence of the wild-typeEV-D68 has a sequence identity of at least 70%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% to a nucleotide sequence as shown in SEQ ID NO: 1. Incertain preferred embodiments, the cDNA sequence of the wild-type EV-D68is a nucleotide sequence as shown in SEQ ID NO: 1.

In certain preferred embodiments, as compared to the wild-type EV-D68,the modified EV-D68 has one or more modifications selected from thefollowing:

(1) one or more mutations in an untranslated region (e.g., 5′UTR or3′UTR);

(2) an insertion of one or more exogenous nucleic acids;

(3) a deletion or mutation of one or more endogenous genes; and

(4) any combination of the above three items.

In certain preferred embodiments, the modified EV-D68 includes one ormore mutations in the 5′ untranslated region (5′UTR).

In certain preferred embodiments, the modified EV-D68 has a substitutionof all or part of the 5′UTR sequence. In certain preferred embodiments,the internal ribosome entry site (IRES) sequence in the 5′UTR of themodified EV-D68 is replaced with an exogenous IRES sequence, such as theinterior ribosome entry site sequence of human rhinovirus 2 (HRV2). Incertain preferred embodiments, the internal ribosome entry site sequenceof the human rhinovirus 2 (HRV2) is shown in SEQ ID NO: 2.

In certain preferred embodiments, the modified EV-D68 comprises anexogenous nucleic acid.

In certain preferred embodiments, the exogenous nucleic acid encodes acytokine (e.g., a GM-CSF, preferably a human GM-CSF), or an antitumorprotein or polypeptide (e.g., a scFv against PD-1 or PD-L1, preferably ascFv against human PD-1 or PD-L1) In certain preferred embodiments, theexogenous nucleic acid is inserted between the 5′UTR gene and the VP4gene, or between the VP1 gene and the 2A gene of the genome of themodified EV-D68.

In certain preferred embodiments, the exogenous nucleic acid comprises atarget sequence of microRNA (miRNA) (e.g., miR-133 or miR-206) Incertain preferred embodiments, the target sequence of microRNA isinserted in the 3′ untranslated region (3′UTR) of the genome of themodified EV-D68.

In certain preferred embodiments, the exogenous nucleic acid comprises atarget sequence of one or more (e.g., 2, 3, or 4) microRNA as describedabove. In certain preferred embodiments, the exogenous nucleic acidcomprises a target sequence of miR-133 and/or miR-206. In certainpreferred embodiments, the target sequence of miR-133 is shown in SEQ IDNO: 3. In certain preferred embodiments, the target sequence of miR-206is shown in SEQ ID NO: 4.

In certain preferred embodiments, the modified EV-D68 comprises at leastone insertion of the exogenous nucleic acid as described above and/or atleast one mutation in the untranslated region as described above.

In certain preferred embodiments, the genomic sequence of the modifiedEV-D68 has a sequence identity of at least 70%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% to a nucleotide sequence selected from: the nucleotidesequences as shown in SEQ ID NOs: 13-16. In certain preferredembodiments, the genomic sequence of the modified EV-D68 is selectedfrom the nucleotide sequences as shown in any one of SEQ ID NOs: 13-16.

In certain preferred embodiments, the cDNA sequence of the modifiedEV-D68 has a sequence identity of at least 70%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% to a nucleotide sequence selected from: the nucleotidesequences as shown in SEQ ID NOs: 8-11. In certain preferredembodiments, the cDNA sequence of the modified EV-D68 is selected fromthe nucleotide sequences as shown in any one of SEQ ID NOs: 8-11.

In the present invention, the modified EV-D68 can be obtained by reversegenetics technology, and the reverse genetics technology is known in theart, for example, see Yang L S, Li S X, Liu Y J, et al Virus Res, 2015,210: 165-168; Hou W H, Yang L S, Li S X, et al. Virus Res, 2015, 205:41-44; which are incorporated herein by reference in their entirety. Insuch embodiments, the modified EV-D68 is typically obtained by modifyingthe cDNA of wild-type EV-D68 (e.g., insertion of an exogenous nucleicacid, deletion or mutation of an endogenous gene, or mutation in anon-translated region).

In the present invention, the modified EV-D68 may be pretreated toreduce or eliminate the immune response against the virus in a subject,wherein the pretreatment may comprise: packaging the EV-D68 in alipidosome or micelle, and/or using a protease (e.g., chymotrypsin ortrypsin) to remove the capsid protein of the virus to reduce the humoraland/or cellular immunity against the virus in host.

In the present invention, the modified EV-D68 can be serially passagedfor adaptation in tumor cells. In certain preferred embodiments, thetumor cells may be tumor cell lines or tumor cell strains known in theart, or may be tumor cells obtained by surgical resection or clinicalisolation from an individual (e.g., a subject) having a tumor. Incertain preferred embodiments, the modified EV-D68 is serially passagedfor adaptation in tumor cells obtained from an individual (e.g., asubject) having a tumor. In certain preferred embodiments, the tumorcells are obtained by surgical resection or clinical isolation from anindividual (e.g., a subject) having a tumor. In certain preferredembodiments, the method for serial passaging for adaptation comprises aplurality of (e.g., at least 5, at least 10, at least 15, at least 20)cycles consisting of the following processes: 1) infecting a targettumor cell with a virus; 2) harvesting the virus in a supernatant; and3) reinfecting a fresh target tumor cell with the obtained virus.

In certain preferred embodiments, the modified EV-D68 is used to treat atumor in a subject, or to prepare a medicament for treating a tumor in asubject.

In certain preferred embodiments, the tumor includes, but is not limitedto, cervical cancer, ovarian cancer, endometrial cancer, lung cancer,liver cancer, kidney cancer, neuroblastoma, glioma, breast cancer,melanoma, Prostate cancer, bladder cancer, pancreatic cancer, gastriccancer, colorectal cancer, esophageal cancer, thyroid cancer, laryngealcancer, osteosarcoma, hematopoietic malignancy (such as lymphoma orleukemia).

In certain preferred embodiments, the subject is a mammal, such as ahuman.

In certain preferred embodiments, the modified EV-D68 of the presentinvention has the internal ribosome entry site (IRES) sequence in the5′UTR replaced with the internal ribosome entry site sequence of humanrhinovirus 2 (HRV2) compared to wild type EV-D68.

In certain preferred embodiments, the modified EV-D68 further comprisesan exogenous nucleic acid.

In certain preferred embodiments, the exogenous nucleic acid encodes acytokine (eg, GM-CSF, preferably human GM-CSF), or an antitumor proteinor polypeptide (e.g., a scFv against PD-1 or PD-L1, preferably a scFvagainst human PD-1 or PD-L1). In certain preferred embodiments, theexogenous nucleic acid is inserted between the 5′UTR and the VP4 gene,or between the VP1 gene and the 2A gene of the genome of the modifiedEV-D68.

In certain preferred embodiments, the exogenous nucleic acid comprises atarget sequence of microRNA (microRNA, miRNA) (eg, miR-133 or miR-206).In certain preferred embodiments, the target sequence of the microRNA isinserted in the 3′ untranslated region (3′UTR) of the genome of themodified EV-D68.

In certain preferred embodiments, the exogenous nucleic acid includes atarget sequence of one or more (e.g., two, three, or four) microRNAs asdescribed above. In certain preferred embodiments, the exogenous nucleicacid comprises a target sequence of miR-133 and/or miR-206. In certainpreferred embodiments, the target sequence of the miR-133 is shown inSEQ ID NO: 3. In certain preferred embodiments, the target sequence ofthe miR-206 is shown in SEQ ID NO: 4.

In certain preferred embodiments, the modified EV-D68 comprises aninsertion of at least one exogenous nucleic acid as described above.

In certain preferred embodiments, the genomic sequence of the modifiedEV-D68 has at least 70%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to the nucleotide sequence shown in SEQ ID NO: 13. In certainpreferred embodiments, the genomic sequence of the modified EV-D68 is anucleotide sequence as shown in SEQ ID NO: 1:3.

In certain preferred embodiments, the cDNA sequence of the modifiedEV-D68 has at least 70%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to the nucleotide sequence shown in SEQ ID NO: 8. In certainpreferred embodiments, the cDNA sequence of the modified EV-D68 is anucleotide sequence as shown in SEQ ID NO: 8.

In certain preferred embodiments, the modified EV-D68 is used to treat atumor in a subject.

In certain preferred embodiments, the tumor includes, but is not limitedto, cervical cancer, ovarian cancer, endometrial cancer, lung cancer,liver cancer, kidney cancer, neuroblastoma, glioma, breast cancer,melanoma, prostate cancer, bladder cancer, pancreatic cancer, gastriccancer, colorectal cancer, esophageal cancer, thyroid cancer, laryngealcancer, osteosarcoma, hematopoietic malignancy (such as lymphoma orleukemia).

In certain preferred embodiments, the tumor is selected from the groupconsisting of gastric cancer, endometrial cancer, cervical cancer, andthyroid cancer.

In a fifth aspect, the invention provides an isolated nucleic acidmolecule comprising a sequence selected from:

(1) the genomic sequence or cDNA sequence of the modified EV-D68according to the fourth aspect; and (2) a complementary sequence of thegenomic sequence or cDNA sequence.

In certain preferred embodiments, the isolated nucleic acid moleculeconsists of the genomic sequence or cDNA sequence of the modified EV-D68as described above, or the complementary sequence of the genomicsequence or cDNA sequence.

In certain preferred embodiments, the isolated nucleic acid molecule hasthe genomic sequence of the modified EV-D68 as described above. Incertain preferred embodiments, the isolated nucleic acid molecule isRNA. In certain preferred embodiments, the isolated nucleic acidmolecule has the nucleotide sequence as shown in any one of SEQ ID NOs:12-16.

In certain preferred embodiments, the isolated nucleic acid molecule isa vector (e.g. a cloning vector or an expression vector) comprising agenomic sequence or cDNA sequence of EV-D68 or a modified form thereofas described above, or a complementary sequence of the genomic sequenceor cDNA sequence. In certain preferred embodiments, the isolated nucleicacid molecule is a vector (e.g., a cloning vector or an expressionvector) comprising a cDNA sequence of EV-D68 or a modified form thereofas described above, or a complementary sequence of the cDNA sequence. Incertain preferred embodiments, the isolated nucleic acid molecule is avector (e.g., a cloning vector or an expression vector) comprising anucleotide sequence as shown in any one of SEQ ID NOs: 1, 8-11 or acomplementary sequence thereof.

In certain preferred embodiments, the isolated nucleic acid moleculecomprises a complementary sequence of the genomic sequence of themodified EV-D68 as described above. In certain preferred embodiments,the complementary sequence is complementary to a nucleotide sequenceselected from:

(1) a nucleotide sequence as shown in any one of SEQ ID NOs: 13-16; and

(2) a nucleotide sequence having a sequence identity of at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% to a nucleotide sequence as shown inany one of SEQ ID NOs: 13-16.

In certain preferred embodiments, the isolated nucleic acid moleculecomprises the complementary sequence of the cDNA sequence of themodified EV-D68 as described above. In certain preferred embodiments,the complementary sequence is complementary to a nucleotide sequenceselected from:

(1) a nucleotide sequence as shown in any one of SEQ ID NOs: 8-11; and

(2) a nucleotide sequence having a sequence identity of at least 70%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% to a nucleotide sequence as shown inany one of SEQ ID NOs: 8-11.

In certain preferred embodiments, the isolated nucleic acid molecule hasa nucleotide sequence as shown in SEQ ID NO: 13, or the isolated nucleicacid molecule is a vector (e.g., a cloning vector or an expressionvector) comprising a nucleotide sequence as shown in SEQ ID NO: 8 or acomplementary sequence thereof.

In the present invention, the isolated nucleic acid molecule can bedelivered by any means known in the art, for example, a naked nucleicacid molecule (e.g., naked RNA) can be directly injected, or a non-viraldelivery system can be used. The non-viral delivery system can beobtained from a variety of materials well known in the art, including,but not limited to, the materials described in detail in “Yin H, et al.Nat Rev Genet. 2014 August; 15 (8): 541-55,” and “Riley M K, VermerrisW. Nanomaterials (Base1). 2017 Apr. 28; 7(5). Pii: E94.”, which areincorporated herein by reference in their entirety, such as liposomes,inorganic nanoparticles (such as gold nanoparticles), polymers (such asPEG), and so on.

In certain preferred embodiments, the isolated nucleic acid molecule isused to treat a tumor in a subject, or to prepare a medicament fortreating a tumor in a subject.

In certain preferred embodiments, the tumor includes, but is not limitedto, cervical cancer, ovarian cancer, endometrial cancer, lung cancer,liver cancer, kidney cancer, neuroblastoma, glioma, breast cancer,melanoma, prostate cancer, bladder cancer, pancreatic cancer, gastriccancer, colorectal cancer, esophageal cancer, thyroid cancer, laryngealcancer, osteosarcoma, hematopoietic malignancy (such as lymphoma orleukemia).

In certain preferred embodiments, the subject is a mammal, such as ahuman.

In another aspect, the present invention also relates to apharmaceutical composition comprising the modified EV-D68 according tothe fourth aspect, or the isolated nucleic acid molecule according tothe fifth aspect.

In another aspect, the present invention also relates to use of themodified EV-D68 according to the fourth aspect, or the isolated nucleicacid molecule according to the fifth aspect, in treating a tumor in asubject, or in the manufacture of a medicament for treating a tumor in asubject.

In another aspect, the invention also relates to a method for treating atumor, comprising a step of administering to a subject in need thereofan effective amount of the modified EV-D68 as described in the fourthaspect, or the isolated nucleic acid molecule according to the fifthaspect

Definition of Terms

In the present invention, unless otherwise stated, scientific andtechnical terms used herein have meanings commonly understood by thoseskilled in the art. In addition, the laboratory procedures of cellculture, biochemistry, cell biology, nucleic acid chemistry and the likeused herein are all routine steps widely used in the correspondingfields. Meanwhile, in order to better understand the present invention,definitions and explanations of related terms are provided below.

As used herein, the term “enterovirus D68 (EV-D68)” refers to one kindof Enterovirus D of the genus Enteroviruses of Picornaviridae family,the genome of which is a single-stranded positive-sense RNA, consistingof a 5′ non-coding region (5′UTR), an open reading frame (ORF), a 3′non-coding region (3′UTR), and a poly(A) tail; wherein its ORF encodes aprecursor polyprotein, which can be hydrolyzed and cleaved by itsprotease to produce structural proteins VP1 to VP4 and non-structuralproteins 2A, 2B, 2C, 3A, 3B, 3C and 3D. In order to more clearlydescribe the present invention, the nucleic acid sequences in the EV-D68genome corresponding to the above proteins are called VP1 gene, VP2gene, VP3 gene, VP4 gene, 2A gene, 2B gene, 2C gene, 3A gene, 3B gene,3C gene, and 3D gene, respectively. In the present invention, theexpression “enterovirus D68 (EV-D68)” refers to a wild-type EV-D68,which can be isolated from sources in nature and has not beenintentionally modified artificially, examples of which include, but arenot limited to, prototype strains AY426531 (CA62-1) and AY426488(CA62-3), and various clinical isolates (for example, the clinicalisolate described in Example 1 of the present invention). The genomicsequence or cDNA sequence of the wild-type EV-D68 is well known in theart and can be found in various public databases (for example, GenBankdatabase, accession number KM881710).

As used herein, the term “modified form” of a virus refers to a modifiedvirus obtained by modifying a wild-type virus, which retains the desiredactivity (e.g., oncolytic activity) of the wild-type virus. In thepresent invention, a “modified form” of EV-D68 includes, but is notlimited to, a modified EV-D68 virus, the genome sequence of which has asubstitution, insertion, or deletion of one or more nucleotides ascompared to that of the wild-type EV-D68, and at least retains theoncolytic activity of EV-D68.

As used herein, the term “oncolytic virus” refers to a virus capable ofinfecting a tumor cell, replicating in the tumor cell, causing the tumorcell death, lysis, or blocking tumor cell growth. Preferably, the virushas minimal toxic effects on a non-tumor cell.

As used herein, the term “tumor-specific” refers to selectivelyexhibiting a biological function or activity within a tumor cell. Forexample, in the present invention, when the term “tumor specificity” isused to describe the killing selectivity of a virus, it means that thevirus can selectively kill a tumor cell without killing or substantiallykilling a non-tumor cell, or the virus is more effective in killing atumor cell than killing a non-tumor cell.

As used herein, the term “oncolytic activity” primarily includes tumorkilling activity. When describing the oncolytic activity of a virus, theoncolytic activity of the virus can typically be measured by indicatorssuch as the virus' ability to infect a tumor cell, ability to replicatein a tumor cell, and/or ability to kill a tumor cell. The oncolyticactivity of a virus can be measured using any method known in the art.For example, the ability of a virus to infect a tumor cell can beevaluated by measuring the viral dose required to infect a givenpercentage of tumor cells (for example, 50% of the cells); the abilityto replicate in a tumor cell can be evaluated by measuring the growth ofthe virus in the tumor cell; the ability to kill a tumor cell can beevaluated by monitoring cytopathic effect (CPE) or measuring tumor cellactivity.

As used herein, the expression “cDNA sequence of EV-D68” means the DNAform of the viral genomic RNA sequence, which differs from the RNAsequence only in that the ribonucleotides in the RNA sequence arereplaced by corresponding deoxyribonucleotides, for example, uracilribonucleotides (UMP) are replaced by thymine deoxyribonucleotides(dTMP).

As used herein, the term “exogenous nucleic acid” refers to anartificially introduced nucleotide sequence that is foreign to theoriginal sequence. Exogenous nucleic acid includes, but is not limitedto, any gene or nucleotide sequence not found in the viral genome.However, in the present invention, it is particularly preferred that theexogenous nucleic acid is composed of at most 1500, such as at most1200, and at most 1000 nucleotides. In some cases, preferably, theexogenous nucleic acid encodes a protein or polypeptide having antitumorkilling activity, such as a cytokine, or an antitumor protein orpolypeptide; or, the exogenous nucleic acid comprises a target sequenceof microRNA (miRNA). In the present invention, the microRNA ispreferably a microRNA having an expression level in a tumor cellsignificantly lower than that in a normal cell and/or having obvioustissue specificity. Examples of the microRNA include, but are notlimited to, miR-122, miR-192, miR-483, etc., which are specificallyexpressed in liver tissue; miR-1, miR-133a/b, miR-208, etc., which arespecifically expressed in heart; miR-192, miR-196a/b, miR-204, miR-215,etc., which are specifically expressed in kidney tissue; miR-133a/b,miR-206, etc., which are specifically expressed in muscle tissue;miR-124a, miR-125a/b, miR-128a/b, miR-138, etc., which are specificallyexpressed in brain tissue; and miR-34, miR-122a, miR-26a, which areunder-expressed in liver tumor tissue; miR-34, which is 1under-expressed in kidney tumor tissue; miR-143, miR-133a/b, which areunder-expressed in bladder tumor tissue; miR-Let-7, miR-29, which areunder-expressed in lung tumor tissue; and so on (see, for example, RuizA J and Russell S J. MicroRNAs and oncolytic viruses. [J]. Curr OpinVirol, 2015, 13: 40-48; which is incorporated herein by reference in itsentirety).

In the present invention, when the modified EV-D68 comprises the targetsequence of microRNA described above, it is regulated by the microRNA ina cell/tissue in which the microRNA is highly expressed or specificallyexpressed, so that replication of the oncolytic virus is attenuated andeven its killing activity is lost, while in a tumor cell/tissue in whichthe microRNA is under-expressed or even not expressed, the oncolyticvirus can normally replicate and thus kill the tumor cell.

As used herein, the term “cytokine” has a meaning well known to thoseskilled in the art. However, in the present invention, when theoncolytic virus of the present invention is used to treat a tumor, it isparticularly preferred that the cytokine is a cytokine that can be usedfor tumor treatment. Examples of “cytokines” include, but are notlimited to, interleukins (e.g., IL-2, IL-12, and IL-15), interferons(e.g., IFNα, IFNβ, IFNγ), tumor necrosis factors (e.g., TNFα), andcolony-stimulating factors (e.g., GM-CSF), and any combination thereof(see, for example, Ardolino M, Hsu J, Raulet D H. Cytokine treatment incancer immunotherapy [J]. Oncotarget, 2015, 6 (23): 19346-19347).

As used herein, the term “antitumor protein or polypeptide” refers to aprotein or polypeptide having antineoplastic activity, including but notlimited to: (1) proteins or polypeptides having toxicity to cells,capable of inhibiting cell proliferation, or inducing apoptosis,examples thereof include, but are not limited to, thymidine kinase TK(TK/GCV), TRAIL, and FasL (see, for example, Candolfi M, King G D,Muhammad A G, et al. Evaluation of proapototic transgenes to use incombination with Flt3L in an immune-stimulatory gene therapy approachfor Glioblastoma multiforme (GBM) [J]. FASEB J, 2008, 22: 1077.13); (2)proteins or polypeptides having immunotherapeutic effects, examplesthereof include, but are not limited to, single chain antibody (scFv)against cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA-4),against programmed death receptor 1 (anti-PD-1), and against programmeddeath ligand 1 (anti-PDL-1) (see, for example, Nolan E, Savas P,Policheni A N, et al. Combined immune checkpoint blockade as atherapeutic strategy for BRCA1-mutated breast cancer [J]. Science TransMed, 2017, 9: eaal 4922; which is incorporated herein by reference inits entirety); (3) proteins or polypeptides that inhibit tumorangiogenesis, examples thereof include, but are not limited to,single-chain antibody (scFv) against vascular endothelial growth factor(anti-VEGF), VEGF -derived polypeptides (e.g., _(D)(LPR),KSVRGKGKGQKRKRKKSRYK, etc.) and ATN-161 (see, for example, Rosca E V,Koskimaki J E, Rivera C G, et al. Anti-angiogenic peptides for cancertherapeutics [J]. Curr Pharm Biotechnol, 2011, 12 (8): 1101-1116; whichis incorporated herein by reference in its entirety).

As used herein, the term “scFv” refers to a single polypeptide chaincomprising a heavy chain variable region (VH) and a light chain variableregion (VL), wherein the VL and VH are linked by a linker (see, forexample, Bird et al., Science 242: 423-426 (1988); Huston et al., Proc.Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, ThePharmacology of Monoclonal Antibodies, No. Volume 113, edited byRoseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). SuchscFv molecule may have a general structure: NH₂-VL-linker-VH-COOH orNH₂-VH-linker-VL-COOH.

As used herein, the term “identity” refers to the match degree betweentwo polypeptides or between two nucleic acids. When two sequences forcomparison have the same monomer sub-unit of base or amino acid at acertain site (e.g., each of two DNA molecules has an adenine at acertain site, or each of two proteins/polypeptides has a lysine at acertain site), the two molecules are identical at the site. The percentidentity between two sequences is a function of the number of identicalsites shared by the two sequences over the total number of sites forcomparison ×100. For example, if 6 of 10 sites of two sequences arematched, these two sequences have an identity of 60%. For example, DNAsequences: CTGACT and CAGGTT share an identity of 50% (3 of 6 sites arematched). Generally, the comparison of two sequences is conducted in amanner to produce maximum identity. Such alignment can be conducted byfor example using a computer program such as Align program (DNAstar,Inc.) which is based on the method of Needleman, et al. (J. Mol. Biol.48:443-453, 1970). The percentage of identity between two amino acidsequences can also be determined using the algorithm of E. Meyers and W.Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has beenincorporated into the ALIGN program (version 2.0), using a PAM120 weightresidue table, and with a gap length penalty of 12 and a gap penalty of4. In addition, the percentage of identity between two amino acidsequences can be determined by the algorithm of Needleman and Wunsch (J.Mol. Biol. 48:444-453 (1970)) which has been incorporated into the GAPprogram in the GCG software package (available at http://www.gcg.com),using either a Blossum 62 matrix or a PAM250 matrix, and with a gapweight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4,5, or 6.

As used herein, the term “vector” refers to a nucleic acid vehicle intowhich a polynucleotide can be inserted. When a vector enables expressionof a protein encoded by an inserted polynucleotide, the vector isreferred to as an expression vector. A vector can be introduced into ahost cell by transformation, transduction, or transfection, so that thegenetic material elements carried by the vector can be expressed in thehost cell. The vector is well known to those skilled in the art andincludes, but is not limited to: plasmids; phagemids; cosmids;artificial chromosomes, such as yeast artificial chromosomes (YAC),bacterial artificial chromosomes (BAC) or P 1 -derived artificialchromosomes (PAC); bacteriophages such as λ-phage or M13 phage andanimal viruses. Animal viruses that can be used as vectors include, butare not limited to, retroviruses (including lentiviruses), adenoviruses,adeno-associated viruses, herpesviruses (such as herpes simplex virus),poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such asSV40). A vector may contain a variety of elements that controlexpression, including, but not limited to, promoter sequences,transcription initiation sequences, enhancer sequences, elements forselection, and reporter genes. In addition, the vector may contain areplication initiation site.

As used herein, the term “internal ribosome entry site (IRES)” refers toa nucleotide sequence located in a messenger RNA (mRNA) sequence that iscapable of initiating translation without the need for the 5′ capstructure. IRES is usually located in the 5′ untranslated region(5′UTR), but may also be located elsewhere in the mRNA.

As used herein, the term “human rhinovirus 2 (HRV2)” refers to a virusof picornaviridae family, the genomic or cDNA sequence of which is wellknown in the art and can be found in various public databases (e.g.,GenBank database, accession number X02316.1).

As used herein, the expression “a nucleic acid molecule comprising agenomic sequence of EV-D68 or a modified form thereof” or “a nucleicacid molecule comprises a genomic sequence of EV-D68 or a modified formthereof” has the meaning commonly understood by those skilled in theart, that is, when the nucleic acid molecule is DNA, the nucleic acidmolecule comprises a genomic sequence of EV-D68 or a modified formthereof in form of DNA; when the nucleic acid molecule is RNA, thenucleic acid molecule comprises a genomic sequence of EV-D68 or amodified form thereof.

As used herein, the term “pharmaceutically acceptable carrier and/orexcipient” refers to a carrier and/or excipient that ispharmacologically and/or physiologically compatible with the subject andthe active ingredient, which is well known in the art (see, for example,Remington's Pharmaceutical Sciences. Edited by Gennaro A R, 19th ed.Pennsylvania: Mack Publishing Company, 1995), and includes, but is notlimited to: pH adjusting agents, surfactants, ionic strength enhancers,agents to maintain osmotic pressure, agents to delay absorption,diluents, adjuvants, preservatives, stabilizers, etc. For example, pHadjusting agents include, but are not limited to, phosphate bufferedsaline. Surfactants include, but are not limited to, cationic, anionicor non-ionic surfactants, such as Tween-80. Ionic strength enhancersinclude, but are not limited to, sodium chloride. Agents that maintainosmotic pressure include, but are not limited to, sugar, NaCl, and thelike. Agents that delay absorption include, but are not limited to,monostearate and gelatin. Diluents include, but are not limited to,water, aqueous buffers (such as buffered saline), alcohols and polyols(such as glycerol), and the like. Adjuvants include, but are not limitedto, aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants(such as complete Freund's adjuvant), and the like. Preservativesinclude, but are not limited to, various antibacterial and antifungalagents, such as thimerosal, 2-phenoxyethanol, parabens,trichloro-t-butanol, phenol, sorbic acid, and the like. Stabilizers havethe meaning commonly understood by those skilled in the art, which canstabilize the desired activity (such as oncolytic activity) of theactive ingredients in the drug, including but not limited to sodiumglutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch,sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamicacid, glycine), proteins (e.g., dried whey, albumin, or casein) or theirdegradation products (e.g., lactalbumin hydrolysates).

As used herein, the term “treating” refers to treating or curing adisease (e.g., a tumor), delaying the onset of symptoms of a disease(e.g., a tumor), and/or delaying the development of a disease (e.g., atumor).

As used herein, the term “effective amount” refers to an amount that caneffectively achieve the intended purpose. For example, a therapeuticallyeffective amount can be an amount effective or sufficient to treat orcure a disease (e.g., a tumor), delay the onset of symptoms of a disease(e.g., a tumor), and/or delay the development of a disease (e.g., atumor). Such an effective amount can be easily determined by a personskilled in the art or a doctor, and can be related to the intendedpurpose (such as treatment), the general health condition, age, gender,weight of the subject, severity, complications, administration route ofthe disease to be treated. The determination of such an effective amountis well within the capabilities of those skilled in the art.

As used herein, the term “subject” refers to a mammal, such as a primatemammal, such as a human. In certain embodiments, the subject (e.g., ahuman) has a tumor, or is at risk for having a tumor.

The Beneficial Effects of the Present Invention

Compared with the prior art, the technical solution of the presentinvention has at least the following beneficial effects:

The inventors of the present application have found for the first timethat enterovirus D68 (EV-D68) has broad-spectrum tumor-killing activity.Based on this finding, the present invention further provides anEV-D68-based oncolytic virus, which has a broader-spectrum tumor-killingactivity and higher tumor specificity, especially also has a very highkilling effect to hematopoietic malignancy (such as lymphoma orleukemia), thus can be used alone for the treatment of tumors, and canalso be used as a supplementary method of traditional tumor treatment,or as a treatment in the absence of other treatment methods.

The EV-D68 or a modified form thereof of the present invention haslittle or no effect on normal cells, and does not induce an immunogenicresponse against the virus in a subject (for example, a human), and thuscan be safely administered to a subject (for example, a human).Therefore, the EV-D68 or a modified form thereof of the presentinvention has great clinical value.

The embodiments of the present invention will be described in detailbelow with reference to the drawings and examples, but those skilled inthe art will understand that the following drawings and examples areonly used to illustrate the present invention, rather than limiting thescope of the present invention. Various objects and advantageous aspectsof the present invention will become apparent to those skilled in theart from the following detailed description of drawings and thepreferred embodiments.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows photomicrographs of the in vitro killing tests of thewild-type EV-D68 on human umbilical vein endothelial cell line HUVEC,human esophageal cancer cell line TE-1, human thyroid cancer cell linesSW-579 and TT in Example 2, wherein MOCK represents cells that are notinfected with the virus. The results showed that the EV-D68 had asignificant oncolytic effect on human tumor cell lines TE-1, SW-579, andTT after 72 hours of infection at a multiplicity of infection (MOI) of10, but had no effect on HUVEC of human normal cells.

FIG. 2 shows the photos of crystal violet staining of the in vitrokilling tests of the wild-type EV-D68 on human liver cancer cell linesHepG2, SMMC7721, BEL7404, BEL7402, and Huh7, human cervical cancer celllines Hela and Caski, human lung cancer cell lines NCI-H1299 and A549,human foreskin fibroblast cell line human embryonic kidney cell lineHEK-293, and differentiated human liver progenitor cell line HepaRG inExample 2, wherein MOCK represents cells that are not infected with thevirus. The results showed that the EV-D68 had significant oncolyticeffects on human tumor cell lines HepG2, SMMC7721, BEL7404, BEL7402,Huh7, Hela, Caski, NCI-H1299 and A549 after 72 hours of infection atMOIs of 10, 1, and 0.1, but had limited effect on HFF-1, HEK-293 anddifferentiated HepaRG of human normal cells.

FIG. 3 shows an electrophoresis image of four samples of wild-typeEV-D68 virus genomic RNA of the same batch obtained by the in vitrotranscription method in Example 2.

FIG. 4 shows the killing effect of the wild-type EV-D68 virus genomicRNA on human cervical cancer tumor cell line Hela in Example 2. Theresults showed that Hela cells showed obvious CPE after 24 hours oftransfection with EV-D68 genomic RNA, and were almost all lysed to deathby 48 hours.

FIGS. 5A to 5C show the results of in vivo antitumor experiment of thewild-type EV-D68 in Example 3 on human cervical cancer cell line Hela(A), human glioma cell line U118-MG (B), and human lymphoma cell lineRaji (C). The results showed that, in the challenge experimental group,10⁶ TCID50 per tumor mass of EV-D68 were injected intratumorally everythird day. After 5 treatments in total, the growth of tumors formed bysubcutaneous inoculation of Hela, U118-MG, or Raji cells in SCID micesignificantly slowed down and arrested, and the tumors were even lysedand disappeared. In contrast, the tumors of the negative group (CTRL)without treatment of oncolytic virus maintained the normal growth, andtheir tumor volumes are significantly larger than those in the challengegroup.

FIG. 6 shows the results of toxicity detection of EV-D68-WT in BALB/cmice in Example 4. FIG. 6A shows the survival rates and health scores of1-day-old BALB/c mice after challenge with EV-D68 at different doses(10³, 10⁴, 10⁵, 10⁶, and 10⁷ TCID50/mouse) by intraperitoneal injection;FIG. 6B shows the survival rates and health scores of BALB/c mice ofdifferent ages (1-day-old, 2-day-old, 3-day-old, 7-day-old and14-day-old) challenged with a very high dose (10⁷ TCID50/mouse) byintraperitoneal injection. The overall toxicity of EV-D68 to BALB/c micewas relatively weak, and only high doses caused the death of 1-day-oldto 3-day-old BALB/c mice, but had no effect on 4- or more-day-old BALB/cmice, indicating that EV-D68 had good safety in vivo.

SEQUENCE INFORMATION

Information of a part of sequences involved in the present invention isprovided in Table 1 as below.

TABLE 1 Sequence description SEQ ID NO: Description 1 cDNA sequence ofwild type EV-D68 (EV-D68-WT) 2 RNA sequence of the internal ribosomeentry site of human rhinovirus 2 (HRV2) 3 RNA sequence of the targetsequence of miR-133 4 RNA sequence of the target sequence of miR-206 5RNA sequence of tandem sequence of miR-133 target sequence and miR-206target sequence 6 DNA sequence of human granulocyte-macrophagecolony-stimulating factor (GM-CSF) gene 7 DNA sequence of anti-humanprogrammed death receptor 1 single chain antibody (Anti-PD-1 scFv) 8cDNA sequence of the modified form of EV-D68 (EV-D68-HRV2) 9 cDNAsequence of the modified form of EV-D68 (EV-D68-miR133&206T) 10 cDNAsequence of the modified form of EV-D68 (EV-D68-GM-CSF) 11 cDNA sequenceof the modified form of EV-D68 (EV-D68-Anti-PD1) 12 Genomic sequence ofwild-type EV-D68 (EV-D68-WT) 13 Genomic sequence of the modified form ofEV-D68 (EV-D68-HRV2) 14 Genomic sequence of the modified form of EV-D68(EV-D68-miR133 & 206T) 15 Genomic sequence of the modified form ofEV-D68 (EV-D68-GM-CSF) 16 Genomic sequence of the modified form ofEV-D68 (EV-D68-Anti-PD1) 17 DNA sequence of miR-133 target sequence 18DNA sequence of miR-206 target sequence 19 DNA sequence of tandemsequence of miR-133 target sequence and miR-206 target sequence 20 DNAsequence of the internal ribosome entry site sequence of humanrhinovirus 2 (HRV2)

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

The present invention is now be described with reference to thefollowing examples which are intended to illustrate the presentinvention (rather than to limit the present invention).

Unless otherwise specified, the molecular biology experimental methodsand immunoassays used in the present invention were carried outsubstantially by referring to the methods described in J. Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocolsin Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995;restriction enzymes were used under conditions recommended by theproduct manufacturer. If the specific conditions were not indicated inthe examples, the conventional conditions or the conditions recommendedby the manufacturer were used. If the reagents or instruments used werenot specified by the manufacturer, they were all conventional productsthat were commercially available. Those skilled in the art willunderstand that the examples describe the present invention by way ofexamples, and are not intended to limit the scope of protection claimedby the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety.

EXAMPLE 1 Obtainment and Preparation of EV-1368 and its Modified Form

1.1 Isolation of Enterovirus EV-D68 from Patient Clinical Sample

(1) A throat swab of patient was gained from the Center for DiseaseControl and Prevention of Xiamen City, China; African green monkeykidney cells (Vero cells; ATCC® Number: CCL-81™) were was kept by theNational Institute of Diagnostics and Vaccine Development in InfectiousDiseases, Xiamen University, China, and cultured in MEM mediumcontaining 10% fetal bovine serum, as well as glutamine, penicillin andstreptomycin.

(2) Sample processing: the throat swab of patient was sufficientlyagitated in a sample preservation solution to wash off the virus andvirus-containing cells adhering to the swab, and then the samplepreservation solution was subjected to a high speed centrifugation at4000 rpm and 4° C. for 30min;

(3) Inoculation and observation:

A) The Vero cells were plated in a 24-well plate with 1×10⁵ cells/well.The growth medium (MEM medium, containing 10% fetal bovine serum, aswell as glutamine, penicillin and streptomycin) was aspirated, and 1 mLof maintenance medium (MEM medium, containing 2% fetal calf serum, aswell as glutamine, penicillin and streptomycin) was added in each well.Then except the negative control wells, each well was inoculated with 50μL of the sample supernatant, and cultured in an incubator at 37° C., 5%CO₂.

B) The cells were observed under a microscope every day for one week,and the occurrence of specific cytopathic effect (CPE) in the inoculatedwells was recorded.

C) If the enterovirus-specific cytopathic effect appeared in the cellsin the inoculated wells within 7 days, the cells and supernatant werecollected and frozen at −80° C.; if no CPE appeared after 7 days, thecells were subjected to blind passage.

D) If CPE appeared within 6 blind passages, the cells and supernatantwere collected and frozen at −80° C.; If CPE did not appear after 6blind passages, the cells were determined as negative.

(4) Isolation and cloning of viruses:

RT-PCR (Piralla et al., J Clin Microbiol 2015, 53 (5): 1725-1726) andenzyme-linked immunospot method based on specific antibody (Yang et al.,Clin Vaccine Immunol 2014, 21 (3): 312 -320; Hou et al., J Virol Methods2015, 215-216: 56-60) were used to identify the viruses isolated fromthe clinical sample, and EV-D68 positive cultures were selected andsubjected to at least 3 cloning experiments. The virus clones obtainedby the limiting dilution method in each experiment were also identifiedby RT-PCR and ELISPOT, and the EV-D68 positive clones were selected forthe next round of cloning. A single EV-D68 strain with strong growthviability was selected as a candidate oncolytic virus strain.

1.2 Rescued Strain of Enterovirus EV-D68 and its Modified Form Obtainedby Infectious Cloning and Reverse Genetics Technology

This example used wild-type EV-D68 (SEQ ID NO: 1) as an example to showhow to obtain EV-D68 and its modified form for the present inventionthrough reverse genetics technology. The specific method was as follows.

(1) Construction of viral infectious clone: the cDNA sequence ofwild-type enterovirus EV-D68 (named EV-D68-WT) was shown in SEQ ID NO:1, and its genomic RNA sequence was SEQ ID NO: 12; or gene insertion orreplacement based on the cDNA (SEQ ID NO: 1) of enterovirus EV-D68 wasperformed, comprising:

Modified form 1: the internal ribosome entry site sequence of wild-typeEV-D68 was replaced with the internal ribosome entry site sequence ofhuman rhinovirus 2 (which has a DNA sequence shown in SEQ ID NO: 20) toobtain the cDNA (SEQ ID NO: 8) of the recombinant virus (named asEV-D68-HRV2), which has a genomic RNA sequence shown as SEQ ID NO: 13;

Modified form 2: the tandem sequence (which has a DNA sequence shown inSEQ ID NO: 19) of miR-133 target sequence (which has a DNA sequenceshown in SEQ ID NO: 17) and miR-206 target sequence (which has a DNAsequence shown in SEQ ID NO: 18) was inserted between 7293-7294 bp ofthe 3′ untranslated region of the cDNA (SEQ ID NO: 1) of the wild-typeEV-D68, to obtain the cDNA (SEQ ID NO : 9) of the recombinant virus(named EV-D68-miR133&206T) , which has a genomic RNA sequence shown asSEQ ID NO: 14;

Modified form 3: the human granulocyte-macrophage colony-stimulatingfactor (GM-CSF) gene (SEQ ID NO: 6) was inserted between the VP1 geneand 2A gene of the cDNA (SEQ ID NO: 1) of wild-type EV-D68 to obtain thecDNA (SEQ ID NO: 10) of the recombinant virus (named EV-D68-GM-CSF),which has a genomic RNA sequence shown as SEQ ID NO: 15;

Modified form 4: the sequence (SEQ ID NO: 7) encoding the single chainantibody against human programmed death receptor 1 (Anti-PD-1 scFv) wasinserted between the VP1 gene and 2A gene of the cDNA (SEQ ID NO: 1) ofwild-type EV-D68 to obtain the cDNA (SEQ ID NO: 11) of the recombinantvirus (named EV-D68-Anti-PD-1), which has a genomic RNA sequence shownas SEQ ID NO: 16.

Then, the cDNA sequences (SEQ ID NO: 1, 8-11) of the above fiveoncolytic viruses were sent to the gene synthesis company (ShanghaiBiotech Engineering Co., Ltd.) for full gene synthesis, and ligated intothe pSVA plasmid (Hou et al. Virus Res 2015, 205: 41-44; Yang et al.,Virus Res 2015, 210: 165-168) to obtain the infectious cloning plasmidsof enterovirus EV-D68 or modified forms thereof (i.e., EV-D68-WT,EV-D68-HRV2, EV-D68-miR133&206T, EV-D68-GM-CSF and EV-D68-Anti-PD-1).

(2) Plasmid mini-kit and E. coli. DH5α competent cells were purchasedfrom Beijing Tiangen Biochemical Technology Co., Ltd.; Hela cells (ATCC®Number: CCL-2™) and human rhabdomyosarcoma cells (RD cells; ATCC®Number: CCL-136™) were kept by National Institute of Diagnostics andVaccine Development in Infectious Diseases, Xiamen University, China,and were cultured with DMEM and MEM media respectively, in which 10%fetal bovine serum as well as glutamine, penicillin and streptomycinwere added; transfection reagents Lipofactamine2000 and Opti-MEM werepurchased from Thermo Fisher Scientific Company.

(3) The infectious cloning plasmids containing the cDNA sequences of theabove five oncolytic viruses were transformed into E. coli DH5ucompetent cells, the monoclonal strains were picked out and shaken afterthe outgrowth of clones, and the plasmids were extracted using theplasmid mini-kit, and then sent to the company (Shanghai BiotechEngineering Co., Ltd.) for sequencing analysis.

(4) The infectious cloning plasmids with correct sequence and the helperplasmid pAR3126 were co-transfected into the cells to rescue virus (Houet al. Virus Res 2015, 205: 41-44; Yang et al. Virus Res 2015, 210:165-168). Hela cells were first transfected according to theinstructions of the transfection reagent; then observed under amicroscope. When CPE appeared in Hela cells, the cells and culturesupernatant were harvested, and inoculated with RD cells followed bypassaging and culturing, thereby obtaining the candidate strain ofoncolytic virus.

EXAMPLE 2 In Vitro Antitumor Experiment of EV-D68 and Modified FormThereof 2.1 Viruses and Cell Lines as Used

(1) Viruses: this example used EV-D68-WT (SEQ ID NO: 12), EV-D68-HRV2(SEQ ID NO: 13), EV-D68-miR133&206T (SEQ ID NO: 14), EV-D68-GM-CSF (SEQID NO: 15) and EV-D68-Anti-PD-1 (SEQ ID NO: 16) as provided in Example1.

(2) Cell lines: human rhabdomyosarcoma cell RD (ATCC® Number: CCL-136™);human cervical cancer cell lines Hela (ATCC® Number: CCL-2™), SiHa(ATCC® Number: HTB-35™), Caski (ATCC® Number: CRL-1550™) and C-33A(ATCC® Number: HTB-31™); human ovarian cancer cell lines SKOV-3/TR(drug-resistant strain of SKOV-3), SKOV-3 (ATCC® Number: HTB-77™) andCaov3 (ATCC® Number: HTB-75™); human endometrial cancer cell linesHec-1-A (ATCC® Number: HTB-112™), Hec-1-B (ATCC® Number: HTB-113™) andIshikawa (ECACC No. 99040201); human lung cancer cell lines SPC-A-1(CCTCC Deposit Number: GDC050), NCI-H1299 (ATCC® Number: CRL-5803™)NCI-H1417 (ATCC® Number: CRL-5869™), NCI-H1703 (ATCC® Number:CRL-5889™), NCI-H1975 (ATCC® Number: CRL-5908™), A549 (ATCC® Number:CCL-185™), NCI-H661 (ATCC® Number: HTB-183™), EBC-1 (Thermo FisherScientific, Catalog #: 11875101), and DMS114 (ATCC® Number: CRL-2066™);human liver cancer cell lines MHCC97H (purchased from the Institute ofLiver Cancer, Fudan University), C3A (ATCC® Number: CRL-10741™), Hep3B(ATCC® Number: HB-8064™), HepG2 (ATCC® Number: HB-8065™), SMMC7721(purchased from the Basic Medical Cell Center of the Institute of BasicMedical Sciences, Chinese Academy of Medical Sciences, number:3111C0001CCC000087), BEL7402 (CCTCC Deposit Number: GDC035), BEL7404(purchased from the Cell Resource Center, Shanghai Institutes ofBiological Sciences, Chinese Academy of Sciences, number:3131C0001000700064), Huh7 (CCTCC Deposit Number: GDC134), PLC/PRF/5(ATCC® Number: CRL8024™) and SK-Hep-1 (ATCC® Number: HTB-52™); humankidney cancer cell lines A-498 (ATCC® Number: HTB-44™), 786-0 (ATCC®Number: CRL-1932™) and Caki-1 (ATCC® Number: HTB-46™); humanneuroblastoma cell lines SH-SYSY (ATCC® Number: CRL-2266™) and SK-N-BE(2) (ATCC® Number: CRL-2271™); human glioma cell lines U87-MG (ATCC®Number: HTB-14™) and U118-MG (ATCC® Number: HTB-15™); human breastcancer cell lines MCF-7 (ATCC® Number: HTB-22™), BcaP37 (CCTCC DepositNumber: GDC206), BT-474 (ATCC® Number: HTB-20™), MDA-MB-231 (ATCC®Number: CRM-HTB-26™) and MDA-MB-453 (ATCC® Number: HTB-131™); humanmelanoma cell lines A-375 (ATCC® Number: CRL-1619™), SK-MEL-1 (ATCC®Number: HTB-67™) and MeWo (ATCC® Number: HTB-65™); human prostate cancercell lines PC-3 (ATCC® Number: CRL-1435™), LNCap (ATCC® Number:CRL1740™) and DU145 (ATCC® Number: HTB-81™); human bladder cancer celllines J82 (ATCC® Number: HTB-1™) and 5637 (ATCC® Number: HTB-9™); humanpancreatic cancer cell lines Capan-2 (ATCC® Number: HTB-80™), HPAF-2(ATCC® Number: CRL-1997™), and PANC-1 (ATCC® Number: CRL-1469™); humangastric cancer cell lines AGS (ATCC® Number: CRL-1739™), SGC7901 (CCTCCDeposit Number: GDC150), BGC823 (CCTCC Deposit Number: GDC151), andNCI-N87 (ATCC® Number: CRL-5822™); human colorectal cancer cell linesDLD-1 (ATCC® Number: CCL-221™), SW1116 (ATCC® Number: CCL-233™), SW480(ATCC® Number: CCL-228™), HCT-116 (ATCC® Number: CCL247™) and HT-29(ATCC® Number: HTB-38™); human esophageal cancer cell line TE-1(purchased from the Cell Resource Center, Shanghai Institutes ofBiological Sciences, Chinese Academy of Sciences, No.3131C0001000700089); human thyroid cancer SW-579 (ATCC® Number:HTB-107™) and TT (ATCC® Number: CRL-1803™); human laryngeal cancer Hep-2(ATCC® Number: CCL-23™); osteosarcoma 143B (ATCC® Number: CRL-8303™) andU2OS (ATCC® Number: HTB-96™); human lymphoma and leukemia cell linesK562 (ATCC® Number: CCL-243™), U937 (ATCC® Number: CRL-1593.2™), THP-1(ATCC® Number: TIB-202™), Raji (ATCC® Number: CCL-86™), Daudi (ATCC®Number: CCL-213™), Jurkat (ATCC® Number: TIB-152™) and MT-4 (obtainedfrom the National Institutes of Health, USA); human normal cell linesinclude: human embryo lung fibroblast cell line MRC-5 (ATCC® Number:CCL-171™), human embryonic kidney cell line HEK-293 (ATCC® Number:CRL-1573™) human foreskin fibroblast cell line FIFF-1 (ATCC® Number:SCRC-1041™), human skin keratinocyte cell line HaCat (CCTCC DepositNumber: GDC106), human prostate stromal cell line WPMY-1 (ATCC® Number:CRL-2854™), human umbilical vein endothelial cell line HUVEC (ThermoFisher Scientific, Catalog #: C01510C), and differentiated human liverprogenitor cell line HepaRG (with characteristics of primaryhepatocytes; Thermo Fisher Scientific, Catalog #: HPRGC10). The abovecells were kept by National Institute of Diagnostics and VaccineDevelopment in Infectious Diseases, Xiamen University, China. HepaRGcells were cultured in WME medium (added with 1.5% DMSO), AGS and TTwere cultured with F-12K medium, SW-579 was cultured with L-15 medium,SH-SY5Y and SK-N-BE (2) were cultured with DMEM:F12 (1:1) medium, RD,C-33A, EBC-1, J82, SK-Hep-1, SK-MEL-1 and DU145 were cultured with MEMmedium, K562, U937, THP-1, Raji, Daudi, Jurkat, MT-4, 5637, 786-O, TE-1,Caski, NCI-H1417, NCI-H1703, NCI-H1975, NCI-H661, SGC7901, BGC823,DLD-1, SW1116, Hep-2, and LNCap were cultured with RPMI-1640 medium,other cells were cultured with DMEM medium. All the mediums mentionedabove were supplemented with 10% fetal bovine serum, glutamine andpenicillin-streptomycin. All the above cells were cultured under thestandard conditions of 37° C. and 5% CO₂.

2.2 Virus Culture

RD cells were evenly plated on 10 cm cell culture plates, and theculture conditions included MEM medium containing 10% fetal bovine serumand glutamine, penicillin and streptomycin, 37° C., 5% CO₂, andsaturated humidity. When the cell confluence reached 90% or more, thecell culture medium was replaced with serum-free MEM medium, and eachplate was inoculated with 10⁷ TCID50 of EV-D68-WT, EV-D68-HRV2,EV-D68-miR133&206T, EV-D68-GM-CSF or EV-D68-Anti-PD-1, the cultureenvironment was changed to 33° C., 5% CO₂, saturated humidity. After 24hours, the EV-D68 or its modified form proliferated in RD cells andcaused CPE in cells. When more than 90% of the cells turned contractedand rounded, showed increased graininess, and became detached and lysed,the cells and culture supernatants thereof were harvested. Afterfreeze-thawing for three cycles, the culture supernatant was collectedand centrifuged to remove cell debris, wherein the centrifuge conditionswere 4000 rpm, 10 min, 4° C. Finally, the supernatant was filtered witha 0.22 μm disposable filter (Millipore Company) to remove impuritiessuch as cell debris.

2.3 Determination of Virus Titer

The RD cells were plated in a 96-well plate with a cell density of 10⁴cells/well. After the cells adhered, the virus solution obtained inExample 2.2 was diluted 10-fold with serum-free MEM medium from thefirst 10-fold dilution. 50 μl of the dilution of virus was added to thewells with cells. After 7 days, the wells where CPE appeared weremonitored and recorded, followed by calculation using Karber method, inwhich the calculation formula was 1 g^(TCID50)=L−D (S−0.5), L: logarithmof the highest dilution, D: difference between the logarithms ofdilutions, S: sum of proportions of positive wells. The unit of TCID50thus calculated was TCID50/50 μl, which should be converted toTCID50/ml.

2.4 In Vitro Antitumor Experiment of Viruses

Human tumor cells and normal cells were inoculated into 96-well platesat 10⁴ cells/well. After the cells adhered, the medium in each well wasreplaced with the corresponding cell culture medium without serum, andviruses were inoculated at an MOI of 0.1, 1, 10 or 100. Subsequently,CPE of the cells were monitored daily by a microscope.

FIG. 1 shows micrographs of the human umbilical vein endothelial cellline HUVEC, the human esophageal cancer cell line TE-1, and the humanthyroid cancer cell line SW-579 and TT, which were not infected withviruses (negative control group, Mock) or which were treated withEV-D68-WT at MOI=10 for 72 hours. The results showed that after 72 hoursof infection at a multiplicity of infection (MOT) of 10, a significantreduction in the number of the tumor cells, marked shrinking and lysisand the like, were detected in the virus-infected groups; while ascompared to the non-tumor cells in the Mock group, the non-tumor cellsinfected with the viruses showed almost no change in cell morphology.The above results demonstrated that EV-D68 had significant oncolyticeffects on human tumor cell lines TE-1, SW-579 and TT, but did not haveany effect on non-tumor cells HUVEC.

After 72 hours of virus infection and culture, the cell survival ratewas detected using Cell Counting Kit-8 (CCK-8 kit; Shanghai BiyuntianBiotechnology Co., Ltd.) and crystal violet staining method (only foradherent cells), and the specific method was as follows:

(1) Cell survival rate detected by CCK8 method

For adherent cells, the original medium in a 96-well cell culture platewas directly discarded; for suspension cells, the original medium in a96-well cell culture plate was carefully discarded after centrifugation;and then 100 μl of fresh serum-free medium was added per well. 10 μl ofCCK-8 solution was added to each of the wells inoculated with cells, andan equal amount of CCK-8 solution was also added to the blank culturemedium as a negative control, followed by incubation at 37° C. in a cellculture incubator for 0.5-3 hours. The absorbance was detected at 450 nmusing a microplate reader at 0.5, 1, 2, 3 hours, respectively, and thetime point where the absorbance was within a suitable range was selectedas a reference for cell survival rate. The CCK-8 test results ofEV-D68-WT for each kind of cells were shown in Table 2, where “−”indicated that the cell survival rate after virus treatment was notsignificantly different from that of the MOCK group; “+” indicated thatafter virus treatment, the cell number was reduced, the survival ratewas still greater than 50% but was significantly different from that ofthe MOCK group; “++” indicated that the cell survival rate after virustreatment was less than 50%, and was significantly different from thatof the MOCK group.

The calculation of cell survival rate was:

${{Cell\_ survival}{\_ rate}(\%)} = {\frac{\begin{matrix}\left( {{{reading\_ of}{\_ test}{\_ group}} -} \right. \\\left. {{reading\_ of}{\_ negative}{\_ group}} \right)\end{matrix}}{\begin{matrix}\left( {{{reading\_ of}{\_ positive}{\_ group}} -} \right. \\\left. {{reading\_ of}{\_ negaive}{\_ group}} \right)\end{matrix}} \times 100{\%.}}$

(2) Cell survival rate detected by crystal violet staining method (onlyfor adherent cells)

After the cells were infected with viruses for 3 days, the culturesupernatant in the 96-well cell culture plate was discarded, 100 μl ofmethanol was added to each well, followed by fixation in the dark for 15min. Crystal violet powder (Shanghai Biotech Biotechnology Co., Ltd.)was weighed, and formulated as 2% (w/v) crystal violet methanolsolution, which was stored at 4° C. An appropriate amount of 2% crystalviolet methanol solution was taken and formulated with PBS solution toprepare 0.2% crystal violet working solution. After fixation for 15minutes, the methanol fixation solution in the 96-well cell cultureplate was discarded, and 100 μl of the crystal violet working solutionwas added to the plate and staining was performed for 30 min. After thecrystal violet staining solution was discarded, PBS solution was usedfor washing for 3 to 5 times, until the excess staining solution waswashed off, and air-drying was performed. ImmunSpot @S5 UV Analyzer(Cellular Technology Limited, USA) was used for photographing. FIG. 2showed the crystal violet staining results of the human liver cancercell lines HepG2, SMMC7721, BEL7404, BEL7402 and Huh7, the humancervical cancer cell lines Hela and Caski, the human lung cancer celllines NCI-H1299 and A549, the human foreskin fibroblast cell line HFF-1,the human embryonic kidney cell line HEK-293, and the differentiatedhuman hepatic progenitor cell line HepaRG in the control group (MOCK)and in the experimental groups (infected for 72 hours with EV-D68-WT atMOIs of 0.1, 1, and 10, respectively). As shown in the results, after 72hours of infection at MOIs of 10, 1, and 0.1, the tumor cells in theexperimental groups were significantly reduced as compared to thecontrol group (MOCK) without addition of virus; while the number ofnon-tumor cells showed no significant change. The above resultsindicated that the EV-D68-WT had significant oncolytic effects on humantumor cell lines HepG2, SMMC7721, BEL7404, BEL7402, Huh7, Hela, Caski,NCI-H1299 and A549, but had no significant effect on non-tumor celllines HFF-1, HEK-293 and the differentiated HepaRG.

TABLE 2 Results of in vitro antitumor experiment of wild-typeenterovirus EV-D68 Multiplicity of infection MOI Cell Line 0.1 1 10 100RD ++ ++ ++ ++ Hela ++ ++ ++ ++ SiHa − − ++ ++ Caski − + ++ ++ C-33A −++ ++ ++ SKOV-3/TR − − − + SKOV-3 − − ++ ++ Caov3 + ++ ++ ++ Hec-1-A − −− ++ Hec-1-B − + ++ ++ Ishikawa − − ++ ++ SPC-A-1 − + ++ ++ NCI-H1299 −++ ++ ++ NCI-H1417 − − − + NCI-H1703 − − − + NCI-H1975 − ++ ++ ++ A549 +++ ++ ++ NCI-H661 − − + ++ EBC-1 − − + ++ DMS114 ++ ++ ++ ++ MHCC97H +++ ++ ++ C3A ++ ++ ++ ++ Hep3B − + + ++ HepG2 − ++ ++ ++ SMMC7721 + ++++ ++ BEL7402 ++ ++ ++ ++ BEL7404 + ++ ++ ++ Huh7 ++ ++ ++ ++ PLC/PRF/5− + ++ ++ SK-Hep-1 − − + ++ A-498 + ++ ++ ++ 786-O − − + ++ Caki-1 ++ ++++ ++ SH-SY5Y − + ++ ++ SK-N-BE(2) − − − + U87-MG + ++ ++ ++ U118-MG ++++ ++ ++ MCF-7 − − − + BcaP37 − ++ ++ ++ BT-474 − − − + MDA-MB-231 ++ ++++ ++ MDA-MB-453 − − + ++ A-375 − + ++ ++ SK-MEL-1 + ++ ++ ++ MeWo − +++ ++ PC-3 ++ ++ ++ ++ LNCap − + ++ ++ DU145 ++ ++ ++ ++ J82 − + ++ ++5637 − − − + Capan-2 − − + ++ HPAF-2 − + + ++ PANC-1 − ++ ++ ++ AGS −− + ++ SGC7901 − − − + BGC823 − + + ++ NCI-N87 − + ++ ++ DLD-1 − − − +SW1116 + + ++ ++ SW480 − + ++ ++ HCT-116 − − + ++ HT-29 + ++ ++ ++ TE-1− + ++ ++ SW-579 − − ++ ++ TT − − ++ ++ Hep-2 − + ++ ++ 143B − − − +U2OS + + ++ ++ K562 − + + ++ U937 − − + ++ THP-1 + ++ ++ ++ Raji ++ ++++ ++ Daudi ++ ++ ++ ++ Jurkat ++ ++ ++ ++ MT-4 ++ ++ ++ ++ MRC-5 − − +++ HEK-293 − − − − HFF-1 − − + + HaCat − − − − WPMY-1 − − − − HUVEC − −− − HepaRG − − − + Note: “−” indicated that there was no significantdifference in cell survival rate between virus treatment group and MOCKgroup; “+” indicated that after virus treatment, the number of cells wasreduced, the survival rate was greater than 50% but was significantlydifferent from that of MOCK group; “++” indicated that the cell survivalrate after virus treatment was less than 50%, and was significantlydifferent from that of the MOCK group.

It could be known from Table 2 that the wild-type enterovirus EV-D68 hada killing effect on the tested tumor cells, and therefore had abroad-spectrum anti-tumor activity. In particular, the virus hadsignificant killing effects on liver cancer cell lines, glioma celllines, prostate cancer cell lines, leukemia and lymphoma cell lines. Onthe other hand, the virus had little or no toxicity to the non-tumorcell lines tested, except that it was significantly toxic to humanembryonic lung fibroblast MRC-5 at higher MOIs.

In addition, in vitro antitumor experiments of EV-D68-HRV2,EV-D68-miR133&206T, EV-D68-GM-CSF and EV-D68-Anti-PD-1 showed that thefour modified EV-D68s retained the broad-spectrum killing effect of thewild-type enterovirus EV-D68 on the tested tumor cells, andsubstantially retained the significant killing effect on the testedtumor cells of human hepatocellular carcinoma cell line, prostate cancercell line, leukemia and lymphoma cell lines, wherein the CCK-8 testresults of oncolytic effect of the four modified EV-D68s on cervicalcancer cell line Hela, glioma cell line U118-MG, liver cancer cell lineHuh7, prostate cancer cell line PC-3, and lymphoma cell line Raji wereshown in Table 3.

TABLE 3 Results of in vitro antitumor experiment of EV-D68-HRV2,EV-D68-miR133&206T, EV-D68-GM-CSF and EV-D68-Anti-PD-1 Multiplicity ofinfection MOI Cell Lines 0.1 1 10 100 EV-D68-HRV2 Hela + + ++ ++U118-MG + ++ ++ ++ Huh7 ++ ++ ++ ++ PC-3 ++ ++ ++ ++ Raji − + + ++EV-D68-miR133&206T Hela ++ ++ ++ ++ U118-MG ++ ++ ++ ++ Huh7 ++ ++ ++ ++PC-3 ++ ++ ++ ++ Raji ++ ++ ++ ++ EV-D68-GM-CSF Hela ++ ++ ++ ++ U118-MG++ ++ ++ ++ Huh7 ++ ++ ++ ++ PC-3 ++ ++ ++ ++ Raji ++ ++ ++ ++EV-D68-Anti-PD-1 Hela ++ ++ ++ ++ U118-MG ++ ++ ++ ++ Huh7 ++ ++ ++ ++PC-3 ++ ++ ++ ++ Raji ++ ++ ++ ++ Note: “−” indicated that there was nosignificant difference in cell survival rate between virus treatmentgroup and MOCK group; “+” indicated that after virus treatment, thenumber of cells was reduced, the survival rate was greater than 50% butwas significantly different from that of MOCK group; “++” indicated thatthe cell survival rate after virus treatment was less than 50%, and wassignificantly different from that of the MOCK group.

In addition, the inventors unexpectedly found that EV-D68-HRV2 exhibitedsignificantly improved killing activity on some tumors compared toEV-D68-WT, wherein the CCK-8 test results of the oncolytic activity ofthe human gastric cancer cell line AGS, the human endometrial cancercell lines HEC-1-A and Ishikawa, the human cervical cancer cell lineC-33A, and the human thyroid cancer cell line SW579 were shown in Table4.

TABLE 4 Comparison of the results of in vitro oncolytic experiment ofEV-D68-WT and EV-D68-HRV2 on some tumor cells MOI Cell Line 0.01 0.1 110 EV-D68-WT AGS − − − + HEC-1-A − − − − Ishikawa − − − ++ C-33A − − ++++ SW579 − − − ++ EV-D68-HRV2 AGS ++ ++ ++ ++ HEC-1-A − + ++ ++ Ishikawa++ ++ ++ ++ C-33A ++ ++ ++ ++ SW579 ++ ++ ++ ++ Note: “−” indicated thatthere was no significant difference in cell survival rate between virustreatment group and MOCK group; “+” indicated that after virustreatment, the number of cells was reduced, the survival rate wasgreater than 50% but was significantly different from that of MOCKgroup; “++” indicated that the cell survival rate after virus treatmentwas less than 50%, and was significantly different from that of the MOCKgroup.

2.5 Serial Passaging of EV-D68 for Adaptation

In this example, EV-D68 was serially passaged for adaptation in acertain type of tumor cell to obtain a virus strain with enhancedkilling activity to the tumor cell.

The wild-type enterovirus EV-D68 was serially passaged for adaptation inthe human cervical cancer cell line SiHa, human ovarian cancer cell lineSKOV-3, human liver cancer cell line SK-hep-1, human pancreatic cancercell line Capan-2, human gastric cancer cell line AGS or humancolorectal cancer cell line HCT-116 on which the oncolytic effect ofEV-D68 was not very significant, and the specific method was as follows:

One kind of the above tumor cells was evenly plated on a 10 cm cellculture plate, and the culture conditions included a corresponding cellculture media containing 10% fetal bovine serum and glutamine,penicillin and streptomycin, 37° C., 5% CO₂, and saturated humidity.When the cell confluence reached 90% or more, the cell culture mediumwas replaced with serum-free cell culture medium, each plate wasinoculated with 10⁷ TCID50 of EV-D68, the culture environment waschanged to 33° C., 5% CO₂, saturated humidity. When EV-D68 proliferatedin tumor cells and caused CPE in the cells (after infection for up to 3days), the cells and their culture supernatant were harvested. Afterfreeze-thawing for three cycles, centrifugation was performed at 4° C.,4000 rpm for 10 min. The centrifugation supernatant was taken and addedonto new tumor cells with a cell confluence of more than 90% to completeone round of virus passage. The passage was repeated for more than 10times, and a part of the virus solution was taken for virus titerdetection in RD cells in each round of passage, and the specific methodreferred to Example 2.3. Generally, the virus replication ability wouldincrease with the generation, and when a relatively high infectioustiter was reached and the virus replication was stable in the tumorcell, the adapted strain of EV-D68 for the tumor cell was obtained.

Subsequently, by the in vitro antitumor experimental method described inExample 2.4, the human tumor cell SiHa, SKOV-3, SK-hep-1, Capan-2, AGS,or HCT-116 was inoculated to a 96-well plate at 10⁴ cells/well. Afterthe cells adhered, the medium in each well was replaced with thecorresponding culture medium free of serum, followed by incubation at37° C. for 30 min, and then the serially passaged EV-D68 virus strains(viral titers of which were detected on RD cells) adapted for each ofthe above kinds of cells at MOIs of 0.1, 1, 10, and 100 were inoculated.Subsequently, CPE of the cells were monitored daily by a microscope, andthe cell survival rate was detected using CCK-8 method 72 hours afterthe infection and culture of viruses.

The results were shown in Table 5, in which after serial passaging ofthe wild-type enterovirus EV-D68 in a certain kind of tumor cells onwhich EV-D68 had poor oncolytic effect, the killing activity thereof onthe tumor cells was significantly enhanced, indicating that the serialpassaging method could be used to obtain an EV-D68 adapted strain withenhanced oncolytic effect on the tumor cells.

TABLE 5 Results of in vitro killing experiment of EV-D68 on a tumor cellafter serial passaging for adaptation in the tumor cell Cell Line 0.1 110 100 SiHa + ++ ++ ++ SKOV-3 − ++ ++ ++ SK-hep-1 − ++ ++ ++ Capan-2 − +++ ++ AGS + + ++ ++ HCT-116 − + ++ ++ Note: “−” indicated that there wasno significant difference in cell survival rate between virus treatmentgroup and MOCK group; “+” indicated that after virus treatment, thenumber of cells was reduced, the survival rate was greater than 50% butwas significantly different from that of MOCK group; “++” indicated thatthe cell survival rate after virus treatment was less than 50%, and wassignificantly different from that of the MOCK group.

2.6 Evaluation of Oncolytic Effect of Genomic RNA of EV-D68

In this example, a large amount of infectious live viruses of EV-D68could be produced by transfecting the purified genomic RNA of EV-D68into a certain kind of tumor cells, and thus kill the tumor cells.

The viral genomic RNA was first obtained by in vitro transcription, andthis method could be found in, for example, Hadac E M, Kelly E J andRussell S J. Mol Ther, 2011, 19(6): 1041-1047. Specifically, theinfectious cloning plasmid of wild-type EV-D68 obtained in Example 1 waslinearized, and the linearized plasmid was used as a template for invitro transcription using MEGAscript™ T7 Transcription Kit (ThermoFisher Scientific, AM1333) so as to produce a large amount of viral RNA.And the obtained viral RNA was purified using MEGAclear™ TranscriptionClean-Up Kit (Thermo Fisher Scientific, AM1908) for next use. The RNAelectropherograms of 4 parallel samples were shown in FIG. 3.

Subsequently, according to the method of the in vitro antitumorexperiment described in Example 2.4, the human cervical cancer tumorcell line Hela was inoculated to a 24-well plate at 10⁵ cells/well.After the cells adhered, the medium in each well was replaced with acorresponding cell culture medium free of serum, followed by incubationat 37° C. for 30 min. Then Hela cells were transfected with purifiedvirus RNA at 1 μg per well using transfection reagent Lipofectamine®2000 (Thermo Fisher Scientific, 11668019), and the negative controlgroup was transfected with irrelevant RNA nucleic acid molecules.Subsequently, CPE of the cells were monitored daily by a microscope.

The results showed that CPE began to appear in the Hela cellstransfected with genomic RNA of EV-D68 about 8 hours after transfection,and then the cytopathy gradually increased. After 48 hours, the survivalrate was measured using the CCK8 method, the Hela cells had almost alldied and lysed. And the micrographs of Hela cells at 0, 24 and 48 hoursafter infection were shown in FIG. 4. The culture supernatant wasinoculated into new Hela cells and CPE was quickly produced. The resultsindicated that the direct administration with the nucleic acid of EV-D68also had good killing activity and could be used to treat tumors.

EXAMPLE 3 In Vivo Antitumor Experiments of Enterovirus EV-D68 and itsModified Forms 3.1 Viruses, Cell Lines and Experimental Animals

(1) Viruses: the EV-D68-WT (SEQ ID NO: 12), EV-D68-HRV2 (SEQ ID NO: 13),EV-D68-miR133&206T (SEQ ID NO: 14), EV-D68-GM-CSF (SEQ ID NO: 15) andEV-D68-Anti-PD-1 (SEQ ID NO: 16) provided in Example 1 were used in thisexample. The methods of virus culture and virus titer measurement couldbe seen in Examples 2.2 and 2.3, respectively.

(2) Cell lines: human cervical cancer cell line Hela (ATCC® Number:CCL-2™), glioma cell line U118-MG (ATCC® Number: HTB-15TH), and lymphomacell line Raji (ATCC® Number: CCL-86™). Except that Raji was culturedwith RPMI-1640 medium, the other Hela and U118-MG were all cultured withDMEM medium. These mediums were all supplemented with 10% fetal bovineserum, glutamine and penicillin-streptomycin. All the above cells werecultured under the standard conditions of 37° C. and 5% CO₂.

(3) Experimental animals: female C.B17 SCID mice aged 6-8 weeks werefrom Shanghai Slark Experimental Animal Co., Ltd.; the mice were raisedunder SPF conditions, according to the protocol approved by theExperimental Animal Center and Ethics Committee of Xiamen University.

3.2 In Vivo Antitumor Experiments of the Virus

The tumor cells used for subcutaneous tumor formation in SCID mice weredigested with 0.01% trypsin, and then resuspended into a single-cellsuspension using a cell culture medium containing 10% fetal bovineserum. The cell density of the suspension was counted. The cells wereprecipitated by centrifugation under 1000 g for 3 min, and then thecells were resuspended with an appropriate volume of PBS to reach aconcentration of about 10⁶-10⁷ cells/100 μl PBS. The tumor cells weresubcutaneously inoculated in the back of SCID mice at 10⁶-10⁷ cells/100μl PBS/site with a syringe. When the tumor cells grew into a tumor massof about 100 mm³ under the skin of SCID mice after about 14-21 days, thetumor-bearing SCID mice were randomly divided into experimental groups(administrated with EV-D68-WT, EV-D68-HRV2, EV-D68-miR133&206T,EV-D68-GM-CSF or EV-D68-Anti-PD-1) and negative control group, with 4mice (n=4) in each group. Oncolytic virus (EV-D68-WT, EV-D68-HRV2,EV-D68-miR133&2061, EV-D68-GM-CSF or EV-D68-Anti-PD-1) at 10⁶ TCID50/100μl serum-free medium/tumor mass or equivalent amount of serum-freemedium were intratumorally injected every two days, for a total of 5treatments. The tumor size was measured with a vernier caliper andrecorded every two days, and the method for calculating the tumor sizewas:

Tumor size (mm³)=tumor length value×(tumor width value)²/2.

The treatment results of EV-D68-WT for the above three tumors were shownin FIGS. 5A-5C. The results showed that after the challenge ofEV-D68-WT, the growth of the three tested tumors of Hela (A), U118-MG(B) and Raji (C) gradually slowed down and arrested, and the tumors wereeven lysed and disappeared; by contrast, the tumors of the negativegroup (CTRL) maintained the normal growth, and their tumor sizes weresignificantly larger than those of the experimental groups.

Table 6 showed the results obtained after a treatment of the Raji tumormodel with EV-D68-WT, EV-D68-HRV2, EV-D68-miR133&206T, EV-D68-GM-CSF orEV-D68-Anti-PD-1 for 10 days. The results showed that the tumor volumeswere significantly reduced after treatment with EV-D68-WT, EV-D68-HRV2,EV-D68-miR133&206T, EV-D68-GM-CSF, and EV-D68-Anti-PD as compared withthe negative control group that was not treated with oncolytic virus,wherein similar reductions in tumor volume were detected after treatmentwith 4 oncolytic viruses EV-D68-WT, EV-D68-miR133&206T, EV-D68-GM-CSFand EV-D68-Anti-PD-1. The above results indicated that all of EV-D68-WT,EV-D68-HRV2, EV-D68-miR133&206T, EV-D68-GM-CSF and EV-D68-Anti-PD-1showed remarkable and favorable antitumor activity in vivo.

TABLE 6 Results of in vivo anti-tumor experiments of EV-D68- WT,EV-D68-HRV2, EV-D68-miR133&206T, EV-D68-GM-CSF and EV-D68-Anti-PD-1 onhuman lymphoma cell line Raji In vivo oncolytic effect Oncolytic viruson Raji after 10 days of treatment EV-D68-WT ++ EV-D68-HRV2 +EV-D68-miR133&206T ++ EV-D68-GM-CSF ++ EV-D68-Anti-PD-1 ++ Note: “+”indicated that after treatment, the tumor volume reducedand was greaterthan 50% of the negative control group, but was significantly differentfrom that of the negative control group; “++” indicated that the tumorvolume reduced to less than 50% of the negative control group aftertreatment, and was significantly different from that of the negativecontrol group.

EXAMPLE 4 Safety Evaluation of Oncolytic Virus 4.1 Viruses andLaboratory Animals Used

(1) Virus: the EV-D68-WT (SEQ ID NO: 12) provided in Example 1 was usedin this example. The methods for virus culture and virus titermeasurement could refer to Examples 2.2 and 2.3, respectively.

(2) Experimental animals: BALB/c pregnant mice were from Shanghai SlarkExperimental Animal Co., Ltd.; according to the protocol approved by theExperimental Animal Center and Ethics Committee of Xiamen University,the mice were raised under clean conditions, and then the 1-day-old,2-day-old, 3-day-old, 7-day-old and 14-day-old mice produced by theBALB/c pregnant mice were used for in vivo virulence evaluation ofEV-D68.

4.2 Evaluation of the Safety of the Virus in Mice

(1) BALB/c suckling mice aged 1 day were selected for challenge withEV-D68-WT by intraperitoneal injection, and the titer doses forchallenge were 10³, 10⁴, 10⁵, 10⁶, or 10⁷ TCID50/mouse. Then thesurvival rates and health scores for the BALB/c mice challenged withdifferent doses were recorded daily, wherein the evaluation criteria ofthe health score were: score of 5, represents dying or died; score of 4represents severe limb paralysis; score of 3 represents weakness or milddeformity of limb; score of 2 represents wasting; score of 1 representslethargy, piloerection, and trembling; and score of 0 representshealthy.

The results were shown in FIG. 6A. Within 20 days after challenge, allmice in the group with extremely high-dose of 10⁷ TCID50 became ill anddied within 1 week; 80% of the mice in the group with high-dose of 10⁶TCID50 eventually survived and only few mice became ill and died; inaddition, no morbidity and death occurred in the mice of the challengegroups with other doses.

(2) The 1-day-old, 2-day-old, 3-day-old, 7-day-old and 14-day-old BALB/cmice were injected with EV-D68-WT at an extremely high dose of 10⁷TCID50/mouse, and then the survival rates and health scores for theBALB/c with different ages in days were recorded daily, wherein theevaluation criteria of the health score were the same as above.

The results were shown in FTG. 6B. Within 20 days after challenge, the1-day-old mice all died within 1 week; the 2-day-old mice resisted in acertain extent to EV-D68 toxicity, and eventually had a survival rate of70%, but with a relatively high incidence of disease and relativelysevere symptoms; the 3-day-old mice were already not vulnerable toEV-D68, and eventually had a survival rate of 90%, and with a lowincidence of disease and mild symptoms; the 4- or more-day-old mice werefully tolerant to the high doses of EV-D68, and no morbidity and deathoccurred.

The above results showed that the EV-D68-WT was less toxic to mice, andwas only lethal to the 1- to 3-day-old BALB/c mice at an extremely highdose of 10⁷ TCID50/mouse, and had no effect on the 4- or more-day-oldmice, thereby indicating good safety in vivo.

Although specific embodiments of the present invention have beendescribed in detail, those skilled in the art will understand thataccording to all the teachings that have been published, variousmodifications and changes can be made to the detail, and these changesare all within the protection scope of the present invention. Theprotection scope of the present invention is given by the appendedclaims and any equivalents thereof.

1. A method of treating a tumor the method comprising administering, toa subject in need thereof, an effective amount of an Enterovirus D68(EV-D68), a modified EV-D68, or an isolated nucleic acid molecule, or amedicament comprising the EV-D68, the modified EV-D68, or the isolatednucleic acid molecule, wherein the isolated nucleic acid moleculecomprises a sequence selected from the group consisting of: (1) agenomic sequence or cDNA sequence of the EV-D68 or the modified EV-D68;and (2) a complementary sequence of the genomic sequence or cDNAsequence.
 2. The method of claim 1, wherein the EV-D68 is a wild-typeEV-D68.
 3. The method of claim 1, wherein as compared to a genome of awild-type EV-D68, a genome of the modified EV-D68 has one or moremodifications selected from the following: (1) a substitution of aninternal ribosome entry site (IRES) sequence in a 5′ untranslated region(5′UTR) with an exogenous IRES sequence; (2) an insertion of one or moreexogenous nucleic acids; (3) a deletion or mutation of one or moreendogenous genes; and (4) any combination of the above three items. 4.The method of claim 3, wherein the exogenous IRES sequence is aninternal ribosome entry site sequence of human rhinovirus 2 (HRV2). 5.The method of claim 3, wherein the one or more exogenous nucleic acidsare selected from the group consisting of a nucleic acid sequenceencoding a cytokine, a nucleic acid sequence encoding an antitumorprotein or polypeptide, and a target sequence of microRNA.
 6. (canceled)7. The method of claim 3, wherein the modified EV-D68 has at least oneof the following characteristics: (1) a genomic sequence of the modifiedEV-D68 has a sequence identity of at least70% to a nucleotide sequenceas shown in SEQ ID NOs: 13-16; and 2) a cDNA sequence of the modifiedEV-D68 has a sequence identity of at least 70% to a nucleotide sequenceas shown in SEQ ID NOs: 8-11.
 8. The method of claim 1, wherein: (1) theisolated nucleic acid molecule consists of a genomic sequence of theEV-D68 or the modified EV-D68; or (2) the isolated nucleic acid moleculeis a vector comprising a cDNA sequence of the EV-D68 or the modifiedEV-D68 or a complementary sequence of the cDNA sequence.
 9. (canceled)10. The method of claim 1, wherein the EV-D68, the modified EV-D68, orthe isolated nucleic acid molecule is administered in combination withan additional pharmaceutically active agent having antitumor activity.11. The method of claim 1, which has at least one of the followingcharacteristics: (1) the tumor is selected from the group consisting ofcervical cancer, ovarian cancer, endometrial cancer, lung cancer, livercancer, kidney cancer, neuroblastoma, glioma, breast cancer, melanoma,prostate cancer, bladder cancer, pancreatic cancer, gastric cancer,colorectal cancer, esophageal cancer, thyroid cancer, laryngeal cancer,osteosarcoma, and hematopoietic malignancy; (2) the subject is a human.12-14. (canceled)
 15. A modified EV-D68, a genome of which has one ormore modifications selected from the following as compared to a genomeof a wild-type EV-D68: (1) a substitution of the internal ribosome entrysite (IRES) sequence in a 5′ untranslated region (5′UTR) with anexogenous IRES sequence; (2) an insertion of one or more exogenousnucleic acids; (3) a deletion or mutation of one or more endogenousgenes; and (4) any combination of the above three items.
 16. Themodified EV-D68 of claim 15, wherein the exogenous IBES sequence is aninternal ribosome entry site sequence of human rhinovirus 2 (HRV2). 17.The modified EV-D68 of claim 15, wherein the one or more exogenousnucleic acids are selected from the group consisting of a nucleic acidsequence encoding a cytokine, a nucleic acid sequence encoding anantitumor protein or polypeptide, and a target sequence of microRNA. 18.(canceled)
 19. The modified EV-D68 of claim 15, wherein the modifiedEV-D68 has at least one of the following characteristics: (1) a genomicsequence of the modified EV-D68 has a sequence identity of at least 70%to a nucleotide sequence as shown in SEQ ID NOs: 13-16; and 2) a cDNAsequence of the modified EV-D68 has a sequence identity of at least 70%,to a nucleotide sequence as shown in SEQ ID NOs: 8-11.
 20. An isolatednucleic acid molecule, which comprises a sequence selected from thegroup consisting of: (1) a genomic sequence or cDNA sequence of themodified EV-D68 of claim 15; and (2) a complementary sequence of thegenomic sequence or cDNA sequence.
 21. The isolated nucleic acidmolecule of claim 20, wherein: (1) the isolated nucleic acid moleculeconsists of a genomic sequence of the EV-D68 or the modified EV-D68: or(2) the isolated nucleic acid molecule is a vector comprising a cDNAsequence of the EV-D68 or the modified EV-D68, or a complementarysequence of the cDNA sequence.
 22. (canceled)
 23. The method of claim 2,wherein the EV-D68 has at least one of the following characteristics:(1) the EV-D68 has a genomic sequence that has a sequence identity of atleast 70% to a nucleotide sequence as shown in SEQ ID NO: 12; and (2)the EV-D68 has a cDNA sequence that has a sequence identity of at least70% to a nucleotide sequence as shown in SEQ ID NO:
 1. 24. The method ofclaim 5, wherein at least one of the following characteristics issatisfied: (i) the cytokine is GM-CSF; (ii) the antitumor protein orpolypeptide is a scFv against PD-1 or PD-L1; (iii) the microRNA isselected from miR-133 and/or miR-206.
 25. The method of claim 11,wherein the hematopoietic malignancy is selected from the groupconsisting of lymphoma and leukemia.
 26. The modified EV-D68 of claim15, wherein the modified EV-D68 has at least one of the followingcharacteristics: (1) a genomic sequence of the wild-type EV-D68 has asequence identity of at least 70% to a nucleotide sequence as shown inSEQ ID NO: 12; and (2) a cDNA sequence of the wild-type EV-D68 has asequence identity of at least 70% to a nucleotide sequence as shown inSEQ ID NO:
 1. 27. The modified EV-D68 of claim 17, wherein at least oneof the following characteristics is satisfied: (i) the cytokine isGM-CSF; (ii) the antitumor protein or polypeptide is a scFv against PD-1or PD-L1; (iii) the microRNA is selected from miR-133 and/or miR-206.